Method for identification and quantification of nucleic acid expression, splice variant, translocation, copy number, or methylation changes

ABSTRACT

The present invention relates to methods and devices for identifying and quantifying, including low abundance, nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicings, exon insertions, exon deletions, intron insertions, or other rearrangement at the genome level and/or methylated nucleotide bases.

This application is a continuation of U.S. patent application Ser. No.15/517,727, filed Apr. 7, 2017, which is a national stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2015/054759, filed, Oct. 8, 2015, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/061,376, filed Oct. 8, 2014,and U.S. Provisional Patent Application Ser. No. 62/103,894, filed Jan.15, 2015, which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for identifying and quantifyingnucleic acid sequence, expression, splice variant, translocation, copynumber, and/or methylation changes using combined nuclease, ligation,and polymerase reactions with carryover prevention.

BACKGROUND OF THE INVENTION

Blood carries oxygen, nutrients, and physiological signals to every cellin the body, while simultaneously providing immunity and protectionagainst outside pathogens. Yet the same ability of blood to spreadsustenance also allows for dissemination of disease, be it cancer cellsmetastasizing to the liver, Ebola virus ravaging the capillaries,Streptococcus pyogenes liquefying flesh, or HIV eluding detection withinthe very CD4 cells that aim to eliminate infections.

The universal propensity of pathogens and cancers alike to spread viathe blood also creates an opportunity for identification and earlydetection—allowing physicians to better treat and manage patient care.The evolution of AIDS treatments went hand-in-hand with improvements innucleic acid diagnostics, from initial reverse-transcription PCR assaysto protect the nations' blood supply, to sequencing drug-resistantvariants, to RT-PCR quantification of viral load to determine treatmentefficacy over time. To date, those infected have not been cured, butsophisticated diagnostic tools have guided treatment, epidemiological,and political decisions to stem this global epidemic.

Cancer is the leading cause of death in developed countries and thesecond leading cause of death in developing countries. Cancer has nowbecome the biggest cause of mortality worldwide, with an estimated 8.2million deaths from cancer in 2012. Cancer cases worldwide are forecastto rise by 75% and reach close to 25 million over the next two decades.A recent report by the world health organization concludes: “(The)Global battle against cancer won't be won with treatment alone.Effective prevention measures (are) urgently needed to prevent (a)cancer crisis”. Detection of early cancer in the blood is the best meansof effective prevention. It will save lives by enabling earlier andbetter treatment, as well as reduce the cost of cancer care.

Plasma or serum from a cancer patient contains nucleic acids releasedfrom cancers cells undergoing abnormal physiological processes. Thesenucleic acids have already demonstrated diagnostic utility (Diaz andBardelli, J Clin Oncol 32: 579-586 (2014); Bettegowda et al., Sci TranslMed 6: 224 (2014); Newman et al., Nat Med 20: 548-554 (2014); Thierry etal., Nat Med 20: 430-435 (2014)). A further source of nucleic acids iswithin circulating tumor cells (CTCs), although early stage and asignificant fraction of localized tumors send out very few to no CTC'sper ml. Normal plasma or serum contains nucleic acids released fromnormal cells undergoing normal physiological processes (i.e. exosomesecretion, apoptosis). There may be additional release of nucleic acidsunder conditions of stress, inflammation, infection, or injury.

The challenge to develop reliable diagnostic and screening tests is todistinguish those markers emanating from the tumor that are indicativeof disease (e.g., early cancer) vs. presence of the same markersemanating from normal tissue (which would lead to a false-positivesignal). There is also a need to balance the number of markers examinedand the cost of the test, with the specificity and sensitivity of theassay. Comprehensive molecular profiling (mRNA, methylation, copynumber, miRNA, mutations) of thousands of tumors by The Cancer GenomeAtlas Consortium (TCGA), has revealed that colorectal tumors are asdifferent from each other as they are from breast, prostrate, or otherepithelial cancers (TCGA “Comprehensive Molecular Characterization ofHuman Colon and Rectal Cancer Nature 487: 330-337 (2014)). Further,those few markers they share in common (e.g., KRAS mutations,) are alsopresent in multiple cancer types, hindering the ability to pinpoint thetissue of origin. For early cancer detection, the nucleic acid assayshould serve primarily as a screening tool, requiring the availabilityof secondary diagnostic follow-up (e.g., colonoscopy for colorectalcancer).

Compounding the biological problem is the need to reliably quantifymutation, promoter methylation, or DNA or RNA copy number from either avery small number of initial cells (i.e. from CTCs), or when the cancersignal is from cell-free DNA (cfDNA) in the blood and diluted by anexcess of nucleic acid arising from normal cells, or inadvertentlyreleased from normal blood cells during sample processing (Mateo et al.,Genome Biol 15: 448 (2014)).

Likewise, an analogous problem of identifying rare target is encounteredwhen using nucleic-acid-based techniques to detect infectious diseasesdirectly in the blood. Briefly, either the pathogen may be present at 1or less colony forming units (cfu)/ml, and/or there are many potentialpathogens and sequence variations responsible for virulence or drugresistance. While these issues are exemplified with cancer, it isrecognized that the solutions are equally applicable to infectiousdiseases.

A continuum of Diagnostic Needs Require a Continuum of Diagnostic Tests.

The majority of current molecular diagnostics efforts in cancer havecentered on:

(i) prognostic and predictive genomics, e.g., identifying inheritedmutations in cancer predisposition genes, such as BrCA1, BrCA2, (Ford etal. Am J Hum Genet 62: 676-689 (1998))

(ii) individualized treatment, e.g., mutations in the EGFR gene guidingpersonalized medicine (Sequist and Lynch, Ann Rev Med, 59: 429-442(2008), and (iii) recurrence monitoring, e.g., detecting emerging KRASmutations in patients developing resistance to drug treatments (Hiley etal., Genome Biol 15: 453 (2014); Amado et al., J Clin Oncol 26:1626-1634 (2008)). Yet, this misses major opportunities in the cancermolecular diagnostics continuum: (i) more frequent screening of thosewith a family history, (ii) screening for detection of early disease,and (iii) monitoring treatment efficacy. To address these three unmetneeds, a new metric for blood-based detection termed “cancer markerload”, analogous to viral load is herein proposed.

DNA sequencing provides the ultimate ability to distinguish all nucleicacid changes associated with disease. However, the process stillrequires multiple up-front sample and template preparation, and is notalways cost-effective. DNA microarrays can provide substantialinformation about multiple sequence variants, such as SNPs or differentRNA expression levels, and are less costly then sequencing; however,they are less suited for obtaining highly quantitative results, nor fordetecting low abundance mutations. On the other end of the spectrum isthe TaqMan™ reaction, which provides real-time quantification of a knowngene, but is less suitable for distinguishing multiple sequence variantsor low abundance mutations.

It is critical to match each unmet diagnostic need with the appropriatediagnostic test—one that combines the divergent goals of achieving bothhigh sensitivity (i.e., low false-negatives) and high specificity (i.e.,low false-positives) at a low cost. For example, direct sequencing ofEGFR exons from a tumor biopsy to determine treatment for non-small celllung cancer (NSCLC) is significantly more accurate and cost effectivethan designing TaqMan™ probes for the over 180 known mutations whosedrug response is already catalogued (Jia et al. Genome Res 23: 1434-1445(2013)). The most sensitive technique for detecting point mutations,BEAMing (Dressman et al., Proc Natl Acad Sci USA 100: 8817-8822 (2003)),rely on prior knowledge of which mutations to look for, and thus arebest suited for monitoring for disease recurrence, rather than for earlydetection. Likewise, to monitor blood levels of Bcr-Abl translocationswhen treating CML patients with Gleevec (Jabbour et al., Cancer 112:2112-2118 (2008)), a simple quantitative reverse-transcription PCR assayis far preferable to sequencing the entire genomic DNA in 1 ml of blood(9 million cells×3 GB=27 million Gb of raw data).

Sequencing 2.1 Gb each of cell-free DNA (cfDNA) isolated from NSCLCpatients was used to provide 10,000-fold coverage on 125 kb of targetedDNA (Kandoth et al. Nature 502: 333-339 (2013)). This approach correctlyidentified mutations present in matched tumors, although only 50% ofstage 1 tumors were covered. The approach has promise for NSCLC, wheresamples average 5 to 20 mutations/Mb, however would not be costeffective for other cancers such as breast and ovarian, that averageless than 1 to 2 mutations per Mb. Current up-front ligation,amplification, and/or capture steps required for highly accuratetargeted deep sequencing are still more complex than multiplexedPCR-TaqMan™ or PCR-LDR assays.

A comprehensive data analysis of over 600 colorectal cancer samples thattakes into account tumor heterogeneity, tumor clusters, andbiological/technical false-positives ranging from 3% to 10% perindividual marker showed that the optimal early detection screen forcolorectal cancer would require at least 5 to 6 positive markers out of24 markers tested (Bacolod et al, Cancer Res 69:723-727 (2009); Tsafriret al. Cancer Res 66: 2129-2137 (2006), Weinstein et al., Nat Genet 45:1113-1120 (2013); Navin N. E. Genome Biol 15: 452 (2014); Hiley et al.,Genome Biol 15: 453 (2014)); Esserman et al. Lancet Oncol 15: e234-242(2014)). Further, marker distribution is biased into different tumorclades, e.g., some tumors are heavily methylated, while others arebarely methylated, and indistinguishable from age-related methylation ofadjacent tissue. Consequently, a multidimensional approach usingcombinations of 3-5 sets of mutation, methylation, miRNA, mRNA,copy-variation, alternative splicing, or translocation markers is neededto obtain sufficient coverage of all different tumor clades. Analogousto non-invasive prenatal screening for trisomy, based on sequencing orperforming ligation detection on random fragments of cfDNA (Benn et al.,Ultrasound Obstet Gynecol. 42(1):15-33 (2013); Chiu et al., Proc NatlAcad Sci USA 105: 20458-20463 (2008); Juneau et al., Fetal Diagn Ther.36(4) (2014)), the actual markers scored in a cancer screen aresecondary to accurate quantification of those positive markers in theplasma.

Technical Challenges of Cancer Diagnostic Test Development.

Diagnostic tests that aim to find very rare or low-abundance mutantsequences face potential false-positive signal arising from: (i)polymerase error in replicating wild-type target, (ii) DNA sequencingerror, (iii) mis-ligation on wild-type target, (iii) target independentPCR product, and (iv) carryover contamination of PCR products arisingfrom a previous positive sample. The profound clinical implications of apositive test result when screening for cancer demand that such a testuse all means possible to virtually eliminate false-positives.

Central to the concept of nucleic acid detection is the selectiveamplification or purification of the desired cancer-specific markersaway from the same or closely similar markers from normal cells. Theseapproaches include: (i) multiple primer binding regions for orthogonalamplification and detection, (ii) affinity selection of CTC's orexosomes, and (iii) spatial dilution of the sample.

The success of PCR-LDR, which uses 4 primer-binding regions to assuresensitivity and specificity, has previously been demonstrated. Desiredregions are amplified using pairs or even tandem pairs of PCR primers,followed by orthogonal nested LDR primer pairs for detection. Oneadvantage of using PCR-LDR is the ability to perform proportional PCRamplification of multiple fragments to enrich for low copy targets, andthen use quantitative LDR to directly identify cancer-specificmutations. Biofire/bioMerieux has developed a similar technology termed“film array”; wherein initial multiplexed PCR reaction products areredistributed into individual wells, and then nested real-time PCRperformed with SYBR Green Dye detection.

Affinity purification of CTC's using antibody or aptamer capture hasbeen demonstrated (Adams et al., J Am Chem Soc 130: 8633-8641 (2008);Dharmasiri et al., Electrophoresis 30: 3289-3300 (2009); Soper et al.Biosens Bioelectron 21: 1932-1942 (2006)). Peptide affinity capture ofexosomes has been reported in the literature. Enrichment of thesetumor-specific fractions from the blood enables copy numberquantification, as well as simplifying screening and verificationassays.

The last approach, spatial dilution of the sample, is employed indigital PCR as well as its close cousin known as BEAMing (Vogelstein andKinzler, Proc Natl Acad Sci USA. 96(16):9236-41 (1999); Dressman et al.,Proc Natl Acad Sci USA 100: 8817-8822 (2003)). The rational for digitalPCR is to overcome the limit of enzymatic discrimination when the samplecomprises very few target molecules containing a known mutation in a1,000 to 10,000-fold excess of wild-type DNA. By diluting input DNA into20,000 or more droplets or beads to distribute less than one molecule oftarget per droplet, the DNA may be amplified via PCR, and then detectedvia probe hybridization or TaqMan™ reaction, giving in essence a 0/1digital score. The approach is currently the most sensitive for findingpoint mutations in plasma, but it does require prior knowledge of themutations being scored, as well as a separate digital dilution for eachmutation, which would deplete the entire sample to score just a fewmutations.

Real-Time PCR & Microfluidic Instrumentation

A number of PCR assays/microfabricated devices have been designed forrapid detection of pathogens and disease-associated translocations andmutations. Each assay/hardware combination has particular strengths, butwhen combined with the real world problem of multidimensional andmultiplexed markers required for cancer detection, the flexibility ofPCR-LDR with microfluidics provides certain advantages.

Instrumentation, assay design, and microfluidic architecture need to beseamlessly integrated. Some PCR instrumentation use real-timefluorescence or end-point fluorescence to quantify initial templatemolecules by cycling chambers, wells, or droplets through differenttemperatures. Yet other instrumentation comprises addressablemicrofluidic plates for real-time PCR detection. However the high costof both the instruments and consumables has limited the widespread useof these machines for clinical applications.

In a different architecture, termed continuous-flow PCR, the reactionmix moves through channels that are neatly arranged in a radiatorpattern, and flow over heating elements that are at fixed temperatures.This architecture allows the entire amplification reaction to becompleted in a few minutes, and is ideal for capillary separation andreadout. For ligase detection reactions, the readout may be achieved bytaking advantage of LDR-FRET or electronic detection. In LDR-FRET, oneprimer has a donor, the other has an acceptor group, and after ligationthey form a hairpin. This allows for counting single ligation events toobtain highly quantitative readouts of input DNA copy number.Alternatively, by appending gold-nanoparticles on each primer, theligation product will contain two nano-particles, and these may bedistinguished using electronic readout.

In considering various degrees of automation, the approach describedherein is guided by the principles of “modularity” and “scalability”.Firstly, the process should be separated into modular steps that mayinitially be optimized on separate instruments. For example, the devicemay be comprised of a first module for purification of DNA from plasmacfDNA as well as RNA from exosomes, a second module for multiplexedreverse transcription and/or limited amplification of various targets,and a third module for generating and detecting ligation products. Sucha modular architecture allows for swapping in improved modules that keeppace with technological developments. For the modularity approach towork, it is critical that products from one module can be movedseamlessly into the next module, without leakage and without worry ofcrossover contamination.

Secondly, the modular design should be amenable to scalable manufacturein high volumes at low cost. The manufacturing costs and howprimers/reagents/samples are deposited into the device must be takeninto consideration.

The present invention is directed at overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by one ormore nucleotides, one or more copy numbers, one or more transcriptsequences, and/or one or more methylated residues. This method involvesproviding a sample potentially containing one or more nucleic acidmolecules containing the target nucleotide sequence differing from thenucleotide sequences in other nucleic acid molecules by one or morenucleotides, one or more copy numbers, one or more transcript sequences,and/or one or more methylated residues, and contacting the sample withone or more enzymes capable of digesting deoxyuracil (dU) containingnucleic acid molecules present in the sample. One or more primaryoligonucleotide primer sets are provided, each primary oligonucleotideprimer set comprising (a) a first primary oligonucleotide primer thatcomprises a nucleotide sequence that is complementary to a sequenceadjacent to the target nucleotide sequence, and (b) a second primaryoligonucleotide primer that comprises a nucleotide sequence that iscomplementary to a portion of an extension product formed from the firstprimary oligonucleotide primer. The contacted sample is blended with theone or more primary oligonucleotide primer sets, a deoxynucleotide mixincluding dUTP, and a DNA polymerase to form a polymerase chain reactionmixture, and the polymerase chain reaction mixture is subjected to oneor more polymerase chain reaction cycles comprising a denaturationtreatment, a hybridization treatment, and an extension treatment,thereby forming primary extension products comprising the targetnucleotide sequence or a complement thereof. The method further involvesblending the primary extension products with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture. Eacholigonucleotide probe set comprises (a) a first oligonucleotide probehaving a target nucleotide sequence-specific portion, and (b) a secondoligonucleotide probe having a target nucleotide sequence-specificportion, and wherein the first and second oligonucleotide probes of aprobe set are configured to hybridize, in a base specific manner, on acomplementary target nucleotide sequence of a primary extension product.The first and second oligonucleotide probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligation reaction mixture, and the ligated productsequences in the sample are detected and distinguished to identify thepresence of one or more nucleic acid molecules containing targetnucleotide sequences differing from nucleotide sequences in othernucleic acid molecules in the sample by one or more nucleotides, one ormore copy numbers, one or more transcript sequences, and/or one or moremethylated residues.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by one ormore nucleotides, one or more copy numbers, one or more transcriptsequences, and/or one or more methylated residues. This method involvesproviding a sample containing one or more nucleic acid moleculespotentially containing the target nucleotide sequence differing from thenucleotide sequences in other nucleic acid molecules by one or morenucleotides, one or more copy numbers, one or more transcript sequences,and/or one or more methylated residues. The method further involvesproviding one or more enzymes capable of digesting deoxyuracil (dU)containing nucleic acid molecules present in the sample, and providingone or more primary oligonucleotide primer sets, each primaryoligonucleotide primer set comprising (a) a first primaryoligonucleotide primer that comprises a nucleotide sequence that iscomplementary to a sequence adjacent to the target nucleotide sequenceand (b) a second primary oligonucleotide primer that comprises anucleotide sequence that is complementary to a portion of an extensionproduct formed from the first primary oligonucleotide primer. The sampleis blended with the one or more primary oligonucleotide primer sets, theone or more enzymes capable of digesting deoxyuracil (dU) containingnucleic acid molecules in the sample, a deoxynucleotide mix includingdUTP, and a DNA polymerase to form a polymerase chain reaction mixture.The polymerase chain reaction mixture is subjected to conditionssuitable for digesting deoxyuracil (dU) containing nucleic acidmolecules present in the polymerase chain reaction mixture, and for oneor more polymerase chain reaction cycles comprising a denaturationtreatment, a hybridization treatment, and an extension treatment,thereby forming primary extension products comprising the targetnucleotide sequence or a complement thereof. The method further involvesblending the primary extension products with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture, whereineach oligonucleotide probe set comprises (a) a first oligonucleotideprobe having a 5′ primer-specific portion and a 3′ target nucleotidesequence-specific portion, and (b) a second oligonucleotide probe havinga 5′ target nucleotide sequence-specific portion and a 3′primer-specific portion. The first and second oligonucleotide probes ofa probe set are configured to hybridize, in a base specific manner, on acomplementary target nucleotide sequence of a primary extension product.The ligation reaction mixture is subjected to one or more ligationreaction cycles whereby the first and second oligonucleotide probes ofthe one or more oligonucleotide probe sets are ligated together to formligated product sequences in the ligation reaction mixture where eachligated product sequence comprises the 5′ primer-specific portion, thetarget-specific portions, and the 3′ primer-specific portion. The methodfurther involves providing one or more secondary oligonucleotide primersets, each secondary oligonucleotide primer set comprising (a) a firstsecondary oligonucleotide primer comprising the same nucleotide sequenceas the 5′ primer-specific portion of the ligated product sequence and(b) a second secondary oligonucleotide primer comprising a nucleotidesequence that is complementary to the 3′ primer-specific portion of theligated product sequence, and blending the ligated product sequences,the one or more secondary oligonucleotide primer sets, the one or moreenzymes capable of digesting deoxyuracil (dU) containing nucleic acidmolecules, a deoxynucleotide mix including dUTP, and a DNA polymerase toform a second polymerase chain reaction mixture. The second polymerasechain reaction mixture is subjected to conditions suitable for digestingdeoxyuracil (dU) containing nucleic acid molecules present in the secondpolymerase chain reaction mixture, and one or more polymerase chainreaction cycles comprising a denaturation treatment, a hybridizationtreatment, and an extension treatment thereby forming secondaryextension products. The secondary extension products are detected anddistinguished in the sample to identify the presence of one or morenucleic acid molecules containing target nucleotide sequences differingfrom nucleotide sequences in other nucleic acid molecules in the sampleby one or more nucleotides, one or more copy numbers, one or moretranscript sequences, and/or one or more methylated residues.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by one ormore nucleotides, one or more copy numbers, one or more transcriptsequences, and/or one or more methylated residues. This method involvesproviding a sample containing one or more nucleic acid moleculespotentially containing the target nucleotide sequence differing from thenucleotide sequences in other nucleic acid molecules by one or morenucleotides, one or more copy numbers, one or more transcript sequences,and/or one or more methylated residues; providing one or more enzymescapable of digesting deoxyuracil (dU) containing nucleic acid moleculespresent in the sample; and providing one or more primary oligonucleotideprimer sets, each primary oligonucleotide primer set comprising (a) afirst primary oligonucleotide primer that comprises a nucleotidesequence that is complementary to a sequence adjacent to the targetnucleotide sequence and (b) a second primary oligonucleotide primer thatcomprises a nucleotide sequence that is complementary to a portion of anextension product formed from the first primary oligonucleotide primer.The method further involves blending the sample, the one or more primaryoligonucleotide primer sets, the one or more enzymes capable ofdigesting deoxyuracil (dU) containing nucleic acid molecules in thesample, a deoxynucleotide mix including dUTP, and a DNA polymerase toform a polymerase chain reaction mixture. The polymerase chain reactionmixture is subjected to conditions suitable for digesting deoxyuracil(dU) containing nucleic acid molecules present in the polymerase chainreaction mixture, and for one or more polymerase chain reaction cyclescomprising a denaturation treatment, a hybridization treatment, and anextension treatment, thereby forming primary extension productscomprising the target nucleotide sequence or a complement thereof. Theprimary extension products are blended with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture, whereineach oligonucleotide probe set comprises (a) a first oligonucleotideprobe having a 5′ portion and a 3′ target nucleotide sequence-specificportion, and (b) a second oligonucleotide probe having a 5′ targetnucleotide sequence-specific portion and a 3′ portion, where the 5′portion of the first oligonucleotide probe of the probe set iscomplementary to a portion of the 3′ portion of the secondoligonucleotide probe, where one probe of the probe set comprises adetectable signal generating moiety, and where the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on a complementary target nucleotide sequence of aprimary extension product. The method further involves subjecting theligation reaction mixture to one or more ligation reaction cycleswhereby the first and second oligonucleotide probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligation reaction mixture where each ligated productsequence comprises the 5′ portion, the target-specific portions, the 3′portion, and the detectable signal generating moiety. The 5′ portion ofthe ligated product sequence is hybridized to its complementary 3′portion and signal from the detectable signal generating moiety that isproduced upon said hybridizing is detected. The ligated productsequences are distinguished in the sample based on said detecting toidentify the presence of one or more nucleic acid molecules containingtarget nucleotide sequences differing from nucleotide sequences in othernucleic acid molecules in the sample by one or more nucleotides, one ormore copy numbers, one or more transcript sequences, and/or one or moremethylated residues.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by one ormore methylated residue. This method involves providing a samplepotentially containing one or more nucleic acid molecules comprising thetarget nucleotide sequence differing from the nucleotide sequences inother nucleic acid molecules by one or more methylated residues andcontacting the sample with one or more enzymes capable of digestingdeoxyuracil (dU) containing nucleic acid molecules present in thesample. The method further involves contacting the sample with one ormore methylation sensitive enzymes to form a restriction enzyme reactionmixture, wherein the one or more methylation sensitive enzyme cleavesnucleic acid molecules in the sample that contain one or moreunmethylated residues within at least one methylation sensitive enzymerecognition sequence. One or more primary oligonucleotide primer setsare provided, each primary oligonucleotide primer set comprising (a)first primary oligonucleotide primer comprising a nucleotide sequencethat is complementary to a region of the target nucleotide sequence thatis upstream of the one or more methylated residues and (b) a secondprimary oligonucleotide primer comprising a nucleotide sequence that isthe same as a region of the target nucleotide sequence that isdownstream of the one or more methylated residues. The restrictionenzyme reaction mixture is blended with the one or more primaryoligonucleotide primer sets, a deoxynucleotide mix including dUTP, and aDNA polymerase to form a primary polymerase chain reaction mixture. Themethod further involves subjecting the primary polymerase chain reactionmixture to one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment, thereby forming primary extension products comprising thetarget nucleotide sequence or a complement thereof. One or moresecondary oligonucleotide primer sets are provided, each secondaryoligonucleotide primer set comprising first and second nestedoligonucleotide primers capable of hybridizing to the primary extensionproducts The primary extension products are blended with the one or moresecondary oligonucleotide primer sets, a deoxynucleotide mix includingdUTP, and a DNA polymerase to form a secondary polymerase chain reactionmixture, and the secondary polymerase chain reaction mixture issubjected to one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming secondary extension products. The secondaryextension products in the sample are detected and distinguished toidentify the presence of one or more nucleic acid molecules containingtarget nucleotide sequences differing from nucleotide sequences in othernucleic acid molecules in the sample by one or more methylated residues.

Another aspect of the present invention is directed to a method foridentifying in a sample, one or more target ribonucleic acid moleculesdiffering in sequence from other ribonucleic acid molecules in thesample due to alternative splicing, alternative transcript, alternativestart site, alternative coding sequence, alternative non-codingsequence, exon insertion, exon deletion, intron insertion,translocation, mutation, or other rearrangement at the genome level.This method involves providing a sample containing one or more targetribonucleic acid molecules potentially containing a sequence differingfrom other ribonucleic acid molecules, and contacting the sample withone or more enzymes capable of digesting dU containing nucleic acidmolecules potentially present in the sample. One or more oligonucleotideprimers are provided, each primer being complementary to the one or moretarget ribonucleic acid molecule. The contacted sample is blended withthe one or more oligonucleotide primers, and a reverse-transcriptase toform a reverse-transcription mixture, and complementary deoxyribonucleicacid (cDNA) molecules are generated in the reverse transcriptionmixture. Each cDNA molecule comprises a nucleotide sequence that iscomplementary to the target ribonucleic acid molecule sequence andcontains dU. The method further involves providing one or moreoligonucleotide primer sets, each primer set comprising (a) a firstoligonucleotide primer comprising a nucleotide sequence that iscomplementary to a portion of a cDNA nucleotide sequence adjacent to thetarget ribonucleic acid molecule sequence complement of the cDNA, and(b) a second oligonucleotide primer comprising a nucleotide sequencethat is complementary to a portion of an extension product formed fromthe first oligonucleotide primer. The reverse transcription mixturecontaining the cDNA molecules is blended with the one or moreoligonucleotide primer sets, and a polymerase to form a polymerasereaction mixture, and the polymerase chain reaction mixture is subjectedto one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming one or more different primary extensionproducts. The method further involves providing one or moreoligonucleotide probe sets. Each probe set comprises (a) a firstoligonucleotide probe having a target sequence-specific portion, and (b)a second oligonucleotide probe having a target sequence-specificportion, wherein the first and second oligonucleotide probes of a probeset are configured to hybridize, in a base specific manner, on acomplementary portion of a primary extension product corresponding tothe target ribonucleic acid molecule sequence. The primary extensionproducts are contacted with a ligase and the one or more oligonucleotideprobe sets to form a ligation reaction mixture and the first and secondprobes of the one or more oligonucleotide probe sets are ligatedtogether to form ligated product sequences in the ligase reactionmixture. The ligated product sequences in the sample are detected anddistinguished thereby identifying the presence of one or more targetribonucleic acid molecules differing in sequence from other ribonucleicacid molecules in the sample due to alternative splicing, alternativetranscript, alternative start site, alternative coding sequence,alternative non-coding sequence, exon insertion, exon deletion, introninsertion, translocation, mutations, or other rearrangement at thegenome level.

Another aspect of the present invention is directed to a method foridentifying in a sample, one or more target ribonucleic acid moleculesdiffering in sequence from other ribonucleic acid molecules in thesample due to alternative splicing, alternative transcript, alternativestart site, alternative coding sequence, alternative non-codingsequence, exon insertion, exon deletion, intron insertion,translocation, mutation, or other rearrangement at the genome level.This method involves providing a sample containing one or more targetribonucleic acid molecules potentially differing in sequence from otherribonucleic acid molecules, and contacting the sample with one or moreenzymes capable of digesting dU containing nucleic acid moleculespotentially present in the sample. One or more oligonucleotide primersis provided, each primer being complementary to the one or more targetribonucleic acid molecules, and the contacted sample is blended with theone or more oligonucleotide primers, a deoxynucleotide mix includingdUTP, and a reverse-transcriptase to form a reverse-transcriptionmixture. Complementary deoxyribonucleic acid (cDNA) molecules aregenerated in the reverse transcription mixture, each cDNA moleculecomprising a nucleotide sequence that is complementary to the targetribonucleic acid molecule and contains dU. The method further involvesproviding one or more oligonucleotide primer sets, each primer setcomprising (a) a first oligonucleotide primer comprising a nucleotidesequence that is complementary to a portion of a cDNA nucleotidesequence adjacent to the target ribonucleic acid molecule sequencecomplement of the cDNA, and (b) a second oligonucleotide primercomprising a nucleotide sequence that is complementary to a portion ofan extension product formed from the first oligonucleotide primer. Thereverse transcription mixture containing the cDNA molecules is blendedwith the one or more oligonucleotide primer sets, a deoxynucleotide mixincluding dUTP, and a polymerase to form a polymerase reaction mixture,and the polymerase chain reaction mixture is subjected to one or morepolymerase chain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby forming oneor more different primary extension products. The method furtherinvolves providing one or more oligonucleotide probe sets, each probeset comprising (a) a first oligonucleotide probe having a 5′primer-specific portion and a 3′ target sequence-specific portion, and(b) a second oligonucleotide probe having a 5′ target sequence-specificportion and a 3′ primer-specific portion, where the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on complementary portions of a primary extensionproduct corresponding to the target ribonucleic acid molecule sequence.The primary extension products are contacted with a ligase and the oneor more oligonucleotide probe sets to form a ligation reaction mixture,and the ligation reaction mixture is subjected to one or more ligationreaction cycles whereby the first and second probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligase reaction mixture, where each ligated productsequence comprises the 5′ primer-specific portion, the target-specificportions, and the 3′ primer-specific portion. The method furtherinvolves providing one or more secondary oligonucleotide primer sets,each secondary oligonucleotide primer set comprising (a) a firstsecondary oligonucleotide primer comprising the same nucleotide sequenceas the 5′ primer-specific portion of the ligated product sequence and(b) a second secondary oligonucleotide primer comprising a nucleotidesequence that is complementary to the 3′ primer-specific portion of theligated product sequence, and blending the ligated product sequences,the one or more secondary oligonucleotide primer sets with one or moreenzymes capable of digesting deoxyuracil (dU) containing nucleic acidmolecules, a deoxynucleotide mix including dUTP, and a DNA polymerase toform a second polymerase chain reaction mixture. The second polymerasechain reaction mixture is subjected to conditions suitable for digestingdeoxyuracil (dU) containing nucleic acid molecules present in the secondpolymerase chain reaction mixture, and one or more polymerase chainreaction cycles comprising a denaturation treatment, a hybridizationtreatment, and an extension treatment thereby forming secondaryextension products. The secondary extension products in the sample aredetected and distinguished thereby identifying the presence of one ormore ribonucleic acid molecules differing in sequence from otherribonucleic acid molecules in the sample due to alternative splicing,alternative transcript, alternative start site, alternative codingsequence, alternative non-coding sequence, exon insertion, exondeletion, intron insertion, translocation, mutation, or otherrearrangement at the genome level.

Another aspect of the present invention is directed to a method foridentifying in a sample, one or more target ribonucleic acid moleculesdiffering in sequence from other ribonucleic acid molecules in thesample due to alternative splicing, alternative transcript, alternativestart site, alternative coding sequence, alternative non-codingsequence, exon insertion, exon deletion, intron insertion,translocation, mutation, or other rearrangement at the genome level.This method involves providing a sample containing one or more targetribonucleic acid molecules potentially differing in sequence from otherribonucleic acid molecules, and contacting the sample with one or moreenzymes capable of digesting dU containing nucleic acid moleculespotentially present in the sample. The method further involves providingone or more oligonucleotide primers, each primer being complementary tothe one or more target ribonucleic acid molecules, and blending thecontacted sample, the one or more oligonucleotide primers, adeoxynucleotide mix including dUTP, and a reverse-transcriptase to forma reverse-transcription mixture. Complementary deoxyribonucleic acid(cDNA) molecules are generated in the reverse transcription mixture,each cDNA molecule comprising a nucleotide sequence that iscomplementary to the target ribonucleic acid molecule and contains dU.The method further involves providing one or more oligonucleotide primersets, each primer set comprising (a) a first oligonucleotide primercomprising a nucleotide sequence that is complementary to a portion of acDNA nucleotide sequence adjacent to the target ribonucleic acidmolecule sequence complement of the cDNA, and (b) a secondoligonucleotide primer comprising a nucleotide sequence that iscomplementary to a portion of an extension product formed from the firstoligonucleotide primer. The reverse transcription mixture containing thecDNA molecules is blended with the one or more oligonucleotide primersets, a deoxynucleotide mix including dUTP, and a polymerase to form apolymerase reaction mixture, and the polymerase chain reaction mixtureis subjected to one or more polymerase chain reaction cycles comprisinga denaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming one or more different primary extensionproducts. The method further involves providing one or moreoligonucleotide probe sets, each probe set comprising (a) a firstoligonucleotide probe having a 5′ portion and a 3′ target nucleotidesequence-specific portion, and (b) a second oligonucleotide probe havinga 5′ target nucleotide sequence-specific portion and a 3′ portion, wherethe 5′ portion of the first oligonucleotide probe of the probe set iscomplementary to a portion of the 3′ portion of the secondoligonucleotide probe, where one probe of the probe set comprises adetectable signal generating moiety, and where the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on complementary portions of a primary extensionproduct corresponding to the target ribonucleic acid molecule sequence.The primary extension products are contacted with a ligase and the oneor more oligonucleotide probe sets to form a ligation reaction mixture,and the ligation reaction mixture is subjected to one or more ligationreaction cycles whereby the first and second probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligase reaction mixture, where each ligated productsequence comprises the 5′ portion, the target-specific portions, the 3′portion, and the detectable signal generating moiety. The 5′ portion ofthe ligated product sequence is hybridized to its complementary 3′portion, and the signal from the detectable signal generating moietythat is produced upon said hybridizing is detected. The ligated productsequences in the sample are detected based on said detecting to identifythe presence of one or more ribonucleic acid molecules differing insequence from other ribonucleic acid molecules in the sample due toalternative splicing, alternative transcript, alternative start site,alternative coding sequence, alternative non-coding sequence, exoninsertion, exon deletion, intron insertion, translocation, mutation, orother rearrangement at the genome level.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more target micro-ribonucleic acid(miRNA) molecules differing in sequence from other miRNA molecules inthe sample by one or more bases. This method involves providing a samplecontaining one or more target miRNA molecules potentially differing insequence from other miRNA molecules in the sample by one or more bases,and contacting the sample with one or more enzymes capable of digestingdU containing nucleic acid molecules potentially present in the sample.One or more oligonucleotide primer sets are provided, each primer setcomprising (a) a first oligonucleotide primer having a 5′ stem-loopportion, a blocking group, an internal primer-specific portion withinthe loop region, and a 3′ nucleotide sequence portion that iscomplementary to a 3′ portion of the target miRNA molecule sequence, (b)a second oligonucleotide primer having a 3′ nucleotide sequence portionthat is complementary to a complement of the 5′ end of the target miRNAmolecule sequence, and a 5′ primer-specific portion, (c) a thirdoligonucleotide primer comprising a nucleotide sequence that is the sameas the internal primer-specific portion of the first oligonucleotideprimer, and (d) a fourth oligonucleotide primer comprising a nucleotidesequence that is the same as the 5′ primer-specific portion of thesecond oligonucleotide primer. The contacted sample is blended with theone or more first oligonucleotide primers of a primer set, adeoxynucleotide mix including dUTP, and a reverse transcriptase to forma reverse transcription reaction mixture. The first oligonucleotideprimer hybridizes to the target miRNA molecule sequence, if present inthe sample, and the reverse transcriptase extends the 3′ end of thehybridized first oligonucleotide primer to generate an extended firstoligonucleotide primer comprising the complement of the target miRNAmolecule sequence. The method further involves blending the reversetranscription reaction mixture with the second, third, and fourtholigonucleotide primers of the primer set to form a polymerase reactionmixture under conditions effective for the one or more secondoligonucleotide primers of a primer set to hybridize to the region ofthe extended first oligonucleotide primer comprising the complement ofthe target miRNA molecule sequence and extend to generate a primaryextension product comprising the 5′ primer-specific portion, anucleotide sequence corresponding to the target miRNA molecule sequence,and the complement of the internal primer-specific portion. Thepolymerase chain reaction mixture is subjected to one or more polymerasechain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby forming aplurality of primary extension products. The method further involvesblending the plurality of primary extension products with a ligase andone or more oligonucleotide probe sets to form a ligation reactionmixture. Each oligonucleotide probe set comprises (a) a firstoligonucleotide probe having a target sequence-specific portion, and (b)a second oligonucleotide probe having a target sequence-specific portionand a portion complementary to a primary extension product, wherein thefirst and second oligonucleotide probes of a probe set are configured tohybridize, in a base specific manner on complementary portions of aprimary extension product corresponding to the target miRNA moleculesequence. The first and second oligonucleotide probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligation reaction mixture, and the ligated productsequences in the sample are detected and distinguished therebyidentifying one or more target miRNA molecules differing in sequencefrom other miRNA molecules in the sample by one or more bases.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more target micro-ribonucleic acid(miRNA) molecules differing in sequence from other miRNA molecules inthe sample by one or more bases. This method involves providing a samplecontaining one or more target miRNA molecules potentially differing insequence from other miRNA molecules in the sample by one or more bases,and contacting the sample with one or more enzymes capable of digestingdU containing nucleic acid molecules potentially present in the sample.The method further involves providing one or more oligonucleotide primersets, each primer set comprising (a) a first oligonucleotide primerhaving a 5′ stem-loop portion, a blocking group, an internalprimer-specific portion within the loop region, and a 3′ nucleotidesequence portion that is complementary to a 3′ portion of the targetmiRNA molecule sequence, (b) a second oligonucleotide primer having a 3′nucleotide sequence portion that is complementary to a complement of the5′ end of the target miRNA molecule sequence, and a 5′ primer-specificportion, (c) a third oligonucleotide primer comprising a nucleotidesequence that is the same as the internal primer-specific portion of thefirst oligonucleotide primer, and (d) a fourth oligonucleotide primercomprising a nucleotide sequence that is the same as the 5′primer-specific portion of the second oligonucleotide primer. Thecontacted sample is blended with the one or more first oligonucleotideprimers of a primer set, a deoxynucleotide mix including dUTP, and areverse transcriptase to form a reverse transcription reaction mixturewhere the first oligonucleotide primer hybridizes to the target miRNAmolecule sequence, if present in the sample, and the reversetranscriptase extends the 3′ end of the hybridized first oligonucleotideprimer to generate an extended first oligonucleotide primer comprisingthe complement of the target miRNA molecule sequence. The reversetranscription reaction mixture is blended with the second, third, andfourth oligonucleotide primers of the primer set to form a polymerasereaction mixture under conditions effective for the one or more secondoligonucleotide primers of a primer set to hybridize to the region ofthe extended first oligonucleotide primer comprising the complement ofthe target miRNA molecule sequence and extend to generate a primaryextension product comprising the 5′ primer-specific portion, anucleotide sequence corresponding to the target miRNA molecule sequence,and the complement of the internal primer-specific portion. Thepolymerase chain reaction mixture is subjected to one or more polymerasechain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby forming aplurality of primary extension products. The plurality of primaryextension products are blended with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture, whereeach oligonucleotide probe set comprises (a) a first oligonucleotideprobe having a 5′ primer-specific portion and a 3′targetsequence-specific portion, and (b) a second oligonucleotide probehaving a 5′ target sequence-specific portion, a portion complementary toa primary extension product, and a 3′ primer-specific portion, and wherethe first and second oligonucleotide probes of a probe set areconfigured to hybridize, in a base specific manner, on complementaryportions of a primary extension product corresponding to the targetmiRNA molecule sequence. The ligation reaction mixture is subjected toone or more ligation reaction cycles whereby the first and secondoligonucleotide probes of the one or more oligonucleotide probe sets areligated together to form ligated product sequences in the ligationreaction mixture wherein each ligated product sequence comprises the 5′primer-specific portion, the target-specific portions, and the 3′primer-specific portion. The method further involves providing one ormore secondary oligonucleotide primer sets, each secondaryoligonucleotide primer set comprising (a) a first secondaryoligonucleotide primer comprising the same nucleotide sequence as the 5′primer-specific portion of the ligated product sequence and (b) a secondsecondary oligonucleotide primer comprising a nucleotide sequence thatis complementary to the 3′ primer-specific portion of the ligatedproduct sequence, and blending the ligated product sequences, the one ormore secondary oligonucleotide primer sets, with one or more enzymescapable of digesting deoxyuracil (dU) containing nucleic acid molecules,a deoxynucleotide mix including dUTP, and a DNA polymerase to form asecond polymerase chain reaction mixture. The second polymerase chainreaction mixture is subjected to conditions suitable for digestingdeoxyuracil (dU) containing nucleic acid molecules present in the secondpolymerase chain reaction mixture, and one or more polymerase chainreaction cycles comprising a denaturation treatment, a hybridizationtreatment, and an extension treatment thereby forming secondaryextension product. The secondary extension products in the sample aredetected and distinguished thereby identifying one or more target miRNAmolecules differing in sequence from other miRNA molecules in the sampleby one or more bases.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more target micro-ribonucleic acid(miRNA) molecules differing in sequence from other miRNA molecules inthe sample by one or more bases. This method involves providing a samplecontaining one or more target miRNA molecules potentially differing insequence from other miRNA molecules in the sample by one or more bases,and contacting the sample with one or more enzymes capable of digestingdU containing nucleic acid molecules potentially present in the sample.The method further involves providing one or more oligonucleotide primersets, each primer set comprising (a) a first oligonucleotide primerhaving a 5′ stem-loop portion, a blocking group, an internalprimer-specific portion within the loop region, and a 3′ nucleotidesequence portion that is complementary to a 3′ portion of the targetmiRNA molecule sequence, (b) a second oligonucleotide primer having a 3′nucleotide sequence portion that is complementary to a complement of the5′ end of the target miRNA molecule sequence, and a 5′ primer-specificportion, (c) a third oligonucleotide primer comprising a nucleotidesequence that is the same as the internal primer-specific portion of thefirst oligonucleotide primer, and (d) a fourth oligonucleotide primercomprising a nucleotide sequence that is the same as the 5′primer-specific portion of the second oligonucleotide primer. Thecontacted sample is blended with the one or more first oligonucleotideprimers of a primer set, a deoxynucleotide mix including dUTP, and areverse transcriptase to form a reverse transcription reaction mixturewherein the first oligonucleotide primer hybridizes to the target miRNAmolecule sequence, if present in the sample, and the reversetranscriptase extends the 3′ end of the hybridized first oligonucleotideprimer to generate an extended first oligonucleotide primer comprisingthe complement of the target miRNA molecule sequence. The reversetranscription reaction mixture is blended with the second, third, andfourth oligonucleotide primers of the primer set to form a polymerasereaction mixture under conditions effective for the one or more secondoligonucleotide primers of a primer set to hybridize to the region ofthe extended first oligonucleotide primer comprising the complement ofthe target miRNA molecule sequence and extend to generate a primaryextension product comprising the 5′ primer-specific portion, anucleotide sequence corresponding to the target miRNA molecule sequence,and the complement of the internal primer-specific portion. The methodfurther involves subjecting the polymerase chain reaction mixture to oneor more polymerase chain reaction cycles comprising a denaturationtreatment, a hybridization treatment, and an extension treatment therebyforming a plurality of primary extension products. The plurality ofprimary extension products are blended with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture, whereineach oligonucleotide probe set comprises (a) a first oligonucleotideprobe having a 5′ portion and a 3′ target nucleotide sequence-specificportion, and (b) a second oligonucleotide probe having a 5′ targetnucleotide sequence-specific portion and a 3′ portion, where the 5′portion of the first oligonucleotide probe of the probe set iscomplementary to a portion of the 3′ portion of the secondoligonucleotide probe, where one probe of the probe set comprises adetectable signal generating moiety, and where the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on complementary portions of a primary extensionproduct corresponding to the target miRNA molecule sequence. Theligation reaction mixture is subjected to one or more ligation reactioncycles whereby the first and second oligonucleotide probes of the one ormore oligonucleotide probe sets are ligated together to form ligatedproduct sequences in the ligation reaction mixture wherein each ligatedproduct sequence comprises the 5′ portion, the target-specific portions,the 3′ portion, and the detectable signal generating moiety. The 5′portion of the ligated product sequence is hybridized to itscomplementary 3′ portion, and signal from the detectable signalgenerating moiety that is produced upon said hybridizing is detected.The ligated product sequences in the sample are distinguished based onsaid detecting to identify the presence one or more target miRNAmolecules differing in sequence from other miRNA molecules in the sampleby one or more bases.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more target micro-ribonucleic acid(miRNA) molecules differing in sequence from other miRNA molecules inthe sample by one or more bases. This method involves providing a samplecontaining one or more target miRNA molecules potentially differing insequence from other miRNA molecules by one or more base differences, andcontacting the sample with one or more enzymes capable of digesting dUcontaining nucleic acid molecules potentially present in the sample. Thecontacted sample is blended with a ligase and a first oligonucleotideprobe comprising a 5′ phosphate, a 5′ stem-loop portion, an internalprimer-specific portion within the loop region, a blocking group, and a3′ nucleotide sequence that is complementary to a 3′ portion of thetarget miRNA molecule sequence to form a ligation reaction. The methodfurther involves ligating the target miRNA molecule sequence at its 3′end to the 5′ phosphate of the first oligonucleotide probe to generate achimeric nucleic acid molecule comprising the target miRNA moleculesequence, if present in the sample, appended to the firstoligonucleotide probe. One or more oligonucleotide primer sets areprovided, each primer set comprising (a) a first oligonucleotide primercomprising a 3′ nucleotide sequence that is complementary to acomplement of the 5′ end of the target miRNA molecule sequence, and a 5′primer-specific portion, (b) a second oligonucleotide primer comprisinga nucleotide sequence that is complementary to the internalprimer-specific portion of the first oligonucleotide probe, and (c) athird oligonucleotide primer comprising a nucleotide sequence that isthe same as the 5′ primer-specific portion of the first oligonucleotideprimer. The chimeric nucleic acid molecule is blended with the one ormore second oligonucleotide primers, a deoxynucleotide mix includingdUTP, and a reverse transcriptase to form a reverse transcriptionreaction mixture, wherein the one or more second oligonucleotide primersof a primer set hybridizes to the internal primer specific portion ofthe chimeric nucleic acid molecule, and extends at its 3′ end togenerate a complement of the chimeric nucleic acid molecule, if presentin the sample. The method further involves blending the reversetranscription reaction mixture with the first and third oligonucleotideprimers of a primer set to form a polymerase reaction mixture, andsubjecting the polymerase chain reaction mixture to one or morepolymerase chain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby formingprimary extension products. The primary extension products comprise the5′ primer-specific portion, a nucleotide sequence corresponding to thetarget miRNA molecule sequence, and the complement of the internalprimer-specific portion. The primary extension products are blended witha ligase and one or more oligonucleotide probe sets to form a ligationreaction mixture. Each oligonucleotide probe set comprises (a) a firstoligonucleotide probe having a target sequence-specific portion, and (b)a second oligonucleotide probe having a target sequence-specific portionand a portion complementary to a primary extension product, wherein thefirst and second oligonucleotide probes of a probe set are configured tohybridize, in a base specific manner, on complementary portions of aprimary extension product corresponding to the target miRNA moleculesequence. The first and second oligonucleotide probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligation reaction mixture, and the ligated productsequences in the sample are detected and distinguished therebyidentifying one or more target miRNA molecules differing in sequencefrom other miRNA molecules in the sample by one or more bases.

Another aspect of the present invention method for identifying, in asample, one or more target micro-ribonucleic acid (miRNA) moleculesdiffering in sequence from other miRNA molecules in the sample by one ormore bases. This method involves providing a sample containing one ormore miRNA molecules potentially differing in sequence from other miRNAmolecules by one or more base differences, and contacting the samplewith one or more enzymes capable of digesting dU containing nucleic acidmolecules potentially present in the sample. The contacted sample isblended with a ligase and a first oligonucleotide probe comprising a 5′phosphate, a 5′ stem-loop portion, an internal primer-specific portionwithin the loop region, a blocking group, and a 3′ nucleotide sequencethat is complementary to a 3′ portion of the target miRNA moleculesequence to form a ligation reaction, and the target miRNA moleculesequence at its 3′ end is ligated to the 5′ phosphate of the firstoligonucleotide probe to generate a chimeric nucleic acid moleculecomprising the target miRNA molecule sequence, if present in the sample,appended to the first oligonucleotide probe. The method further involvesproviding one or more oligonucleotide primer sets, each primer setcomprising (a) a first oligonucleotide primer comprising a 3′ nucleotidesequence that is complementary to a complement of the 5′ end of thetarget miRNA molecule sequence, and a 5′ primer-specific portion, (b) asecond oligonucleotide primer comprising a nucleotide sequence that iscomplementary to the internal primer-specific portion of the firstoligonucleotide probe, and (c) a third oligonucleotide primer comprisinga nucleotide sequence that is the same as the 5′ primer-specific portionof the first oligonucleotide primer. The chimeric nucleic acid moleculeis blended with the one or more second oligonucleotide primers, adeoxynucleotide mix including dUTP, and a reverse transcriptase to forma reverse transcription reaction mixture, where the one or more secondoligonucleotide primers of a primer set hybridizes to the internalprimer specific portion of the chimeric nucleic acid molecule andextends at its 3′ end to generate a complement of the chimeric nucleicacid molecule, if present in the sample. The reverse transcriptionreaction mixture is blended with the first and third oligonucleotideprimers of a primer set to form a polymerase reaction mixture, and thepolymerase chain reaction mixture is subjected to one or more polymerasechain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby formingprimary extension products comprising the 5′ primer-specific portion, anucleotide sequence corresponding to the target miRNA molecule sequence,and the complement of the internal primer-specific portion. The primaryextension products are blended with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture, whereineach oligonucleotide probe set comprises (a) a first oligonucleotideprobe having a 5′ primer-specific portion and a 3′ targetsequence-specific portion, and (b) a second oligonucleotide probe havinga 5′ target sequence-specific portion, a portion complementary to aprimary extension product, and a 3′ primer-specific portion, wherein thefirst and second oligonucleotide probes of a probe set are configured tohybridize, in a base specific manner, on complementary portions of aprimary extension product corresponding to the target miRNA moleculesequence. The ligation reaction mixture is subjected to one or moreligation reaction cycles whereby the first and second oligonucleotideprobes of the one or more oligonucleotide probe sets are ligatedtogether to form ligated product sequences in the ligation reactionmixture, wherein each ligated product sequence comprises the 5′primer-specific portion, the target-specific portions, and the 3′primer-specific portion. The method further involves providing one ormore secondary oligonucleotide primer sets, each secondaryoligonucleotide primer set comprising (a) a first secondaryoligonucleotide primer comprising the same nucleotide sequence as the 5′primer-specific portion of the ligated product sequence and (b) a secondsecondary oligonucleotide primer comprising a nucleotide sequence thatis complementary to the 3′ primer-specific portion of the ligatedproduct sequence. The ligated product sequences are blended with the oneor more secondary oligonucleotide primer sets, one or more enzymescapable of digesting deoxyuracil (dU) containing nucleic acid molecules,a deoxynucleotide mix including dUTP, and a DNA polymerase to form asecond polymerase chain reaction mixture. The second polymerase chainreaction mixture is subjected to conditions suitable for digestingdeoxyuracil (dU) containing nucleic acid molecules present in the secondpolymerase chain reaction mixture, and one or more polymerase chainreaction cycles comprising a denaturation treatment, a hybridizationtreatment, and an extension treatment thereby forming secondaryextension products. The secondary extension products in the sample aredetected and distinguished thereby identifying one or more target miRNAmolecules differing in sequence from other miRNA molecules in the sampleby one or more bases.

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more target micro-ribonucleic acid(miRNA) molecules differing in sequence from other miRNA molecules inthe sample by one or more bases. This method involves providing a samplecontaining one or more miRNA molecules potentially differing in sequencefrom other miRNA molecules by one or more base differences, andcontacting the sample with one or more enzymes capable of digesting dUcontaining nucleic acid molecules potentially present in the sample. Thecontacted sample is blended with a ligase and a first oligonucleotideprobe comprising a 5′ phosphate, a 5′ stem-loop portion, an internalprimer-specific portion within the loop region, a blocking group, and a3′ nucleotide sequence that is complementary to a 3′ portion of thetarget miRNA molecule sequence to form a ligation reaction. The targetmiRNA molecule sequence is ligated at its 3′ end to the 5′ phosphate ofthe first oligonucleotide probe to generate a chimeric nucleic acidmolecule comprising the target miRNA molecule sequence, if present inthe sample, appended to the first oligonucleotide probe. The methodfurther involves providing one or more oligonucleotide primer sets, eachprimer set comprising (a) a first oligonucleotide primer comprising a 3′nucleotide sequence that is complementary to a complement of the 5′ endof the target miRNA molecule sequence, and a 5′ primer-specific portion,(b) a second oligonucleotide primer comprising a nucleotide sequencethat is complementary to the internal primer-specific portion of thefirst oligonucleotide probe, and (c) a third oligonucleotide primercomprising a nucleotide sequence that is the same as the 5′primer-specific portion of the first oligonucleotide primer. Thechimeric nucleic acid molecule is blended with the one or more secondoligonucleotide primers, a deoxynucleotide mix including dUTP, and areverse transcriptase to form a reverse transcription reaction mixture,wherein the one or more second oligonucleotide primers of a primer sethybridizes to the internal primer specific portion of the chimericnucleic acid molecule and extends at its 3′ end to generate a complementof the chimeric nucleic acid molecule, if present in the sample. Thereverse transcription reaction mixture is blended with the first andthird oligonucleotide primers of a primer set to form a polymerasereaction mixture, and the polymerase chain reaction mixture is subjectedto one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming primary extension products comprising the 5′primer-specific portion, a nucleotide sequence corresponding to thetarget miRNA molecule sequence, and the complement of the internalprimer-specific portion. The primary extension products are blended witha ligase and one or more oligonucleotide probe sets to form a ligationreaction mixture, wherein each oligonucleotide probe set comprises (a) afirst oligonucleotide probe having a 5′ portion and a 3′ targetnucleotide sequence-specific portion, and (b) a second oligonucleotideprobe having a 5′ target nucleotide sequence-specific portion and a 3′portion, where the 5′ portion of the first oligonucleotide probe of theprobe set is complementary to a portion of the 3′ portion of the secondoligonucleotide probe, where one probe of the probe set comprises adetectable signal generating moiety, and where the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on complementary portions of a primary extensionproduct corresponding to the target miRNA molecule sequence. Theligation reaction mixture is subjected to one or more ligation reactioncycles whereby the first and second oligonucleotide probes of the one ormore oligonucleotide probe sets are ligated together to form ligatedproduct sequences in the ligation reaction mixture wherein each ligatedproduct sequence comprises the 5′ portion, the target-specific portions,the 3′ portion, and the detectable signal generating moiety. The the 5′portion of the ligated product sequence is hybridized to itscomplementary 3′ portion, and signal from the detectable signalgenerating moiety that is produced upon said hybridizing is detected.The ligated product sequences are distinguished in the sample based onsaid detecting to identify the presence one or more target miRNAmolecules differing in sequence from other miRNA molecules in the sampleby one or more bases.

Another aspect of the present invention is directed to a device forsimultaneously adding liquids to two or more wells in a row and/orcolumn of a microtiter plate. The device has opposed top and bottomsurfaces with the top surface having openings leading into the wells andthe bottom surface defining closed ends of the wells. The devicecomprises a first layer defined by first and second boundaries withmetering chambers extending between the first and second boundaries ofsaid first layer and in fluid communication with one another. The firstlayer is configured to be fitted, in an operative position, proximate tothe microtiter plate with the first boundary of the first layer beingclosest to the top surface of the microtiter plate and each of themetering chambers being in fluid communication with an individual wellin a row and/or column of the microtiter plate. The first layer furthercomprises a filling chamber in fluid communication with one or more ofthe metering chambers. The device comprises a second layer defined byfirst and second boundaries with a filling port extending between thefirst and second boundaries of the second layer. The second layer isconfigured to be fitted, in an operative position, on the first layerwith the first boundary of the second layer adjacent to the secondboundary of the first layer and the filling port being aligned with thefilling chamber. When the first layer, second layer, and microtiterplate are positioned with respect to one another in their operativepositions, liquid entering the device through the filling port will passthrough the input chamber, the metering chambers, and into two or morewells in a row and/or column of the microtiter plate.

Another aspect of the present invention is directed to a method ofadding liquids to two or more wells in a row and/or column of amicrotiter plate having opposed top and bottom surfaces with the topsurface having openings leading into the wells and the bottom surfacedefining closed ends of the wells. This method involves providing adevice comprising a first layer having first and second boundaries withmetering chambers extending between the first and second boundaries ofthe first layer and in fluid communication with one another. The firstlayer of the device is configured to be fitted, in an operativeposition, proximate to the microtiter plate with the first boundaries ofthe first layer being closest to the top surface of the microtiter plateand one of the metering chambers being in fluid communication with anindividual well in a row and/or column of the microtiter plate. Thefirst layer further comprising a filling chamber in fluid communicationwith one or more of said metering chambers. The device comprises asecond layer having first and second boundaries with a filling portextending between the first and second boundaries of the second layer.The second layer is configured to be fitted, in an operative position,on the first layer with the first boundary of the second layer adjacentto the second boundary of the first layer and the filling port beingaligned with the charge chamber. When the first layer, second layer, andmicrotiter plate are positioned with respect to one another in theiroperative positions, liquid entering the device through the filling portwill pass through the filling chamber, the metering chambers, and intotwo or more wells in a row and/or column of said microtiter plate. Themethod further involves filling the device with liquid, and dischargingliquid in the device into two or more wells in a row and/or column ofsaid microtiter plate.

The present invention describes a number of approaches for detectingmutations, expression, splice variant, translocation, copy number,and/or methylation changes in target nucleic acid molecules usingnuclease, ligase and polymerase reactions. The present invention solvesthe problems of carry over prevention, as well as allowing for spatialmultiplexing to provide relative quantification, similar to digital PCR.Such technology may be utilized for non-invasive early detection ofcancer, non-invasive prognosis of cancer, and monitoring for cancerrecurrence from plasma or serum samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a conditional logic tree for an early detection pancancer test based on analysis of a patient's blood sample.

FIG. 2 illustrates a work flow for analysis of DNA, mRNA, and miRNA fromplasma cfDNA, exosomes, and CTC.

FIG. 3 illustrates the generic analysis of DNA, mRNA or miRNA bydistribution of one sample, which has initially been diluted anddistributed across 24 tubes for multiplexed PCR or reverse-transcriptasePCR followed by LDR with tagged probes, into 24×16 rows of a microtiterplate.

FIG. 4 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 3.

FIG. 5 illustrates a hypothetic signal pattern generated from thereal-time detection of each well on a microtiter plate from FIG. 4.

FIG. 6 illustrates the workflow for analysis of DNA from plasma cfDNA orCTC.

FIG. 7 illustrates the analysis of DNA, by distribution of one sample,which has initially been diluted and distributed across 24 tubes formultiplexed PCR or reverse-transcriptase PCR followed by LDR with taggedprobes, into 24×16 rows of a microtiter plate.

FIG. 8 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 7.

FIG. 9 illustrates a hypothetic signal pattern generated from thereal-time detection of each well on a microtiter plate from FIG. 8.

FIG. 10 illustrates a work flow for analysis of mRNA or miRNA fromplasma exosomes and illustrates a work flow for analysis of DNA, mRNA,and miRNA from plasma cfDNA, exosomes and CTC.

FIG. 11 illustrates the analysis of mRNA or miRNA, by distribution ofone sample, which has initially been serially diluted into 24 tubes formultiplexed reverse-transcriptase PCR followed by LDR with taggedprobes, into 24×16 rows of a microtiter plate.

FIG. 12 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 11.

FIG. 13 illustrates a hypothetic signal pattern generated from thereal-time detection of each well on a microtiter plate from FIG. 12.

FIG. 14 illustrates the workflow for the generic analysis of DNA, mRNAor miRNA by distribution of one sample, which has initially been dilutedacross 24 tubes for multiplexed PCR or reverse-transcriptase PCRfollowed by LDR with tagged probes, for streptavidin mediated capture in24×16 rows of a microtiter plate.

FIG. 15 illustrates the generic analysis of DNA, mRNA or miRNA bydistribution of 24 samples, which have initially been diluted anddistributed across 24 tubes for multiplexed PCR or reverse-transcriptasePCR followed by LDR with tagged probes, into 24×16 rows of a microtiterplate.

FIG. 16 illustrates the streptavidin mediated capture of thebiotinylated PCR or RT-PCR amplicons in the wells of a microtiter platefrom FIG. 15.

FIG. 17 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 16.

FIG. 18 illustrates a hypothetic signal pattern generated from thereal-time detection of LDR-FRET in each well on a microtiter plate fromFIG. 17.

FIG. 19 illustrates the workflow for the analysis of DNA by distributionof one sample, which has initially been diluted across 24 tubes formultiplexed PCR followed by LDR with tagged probes, for streptavidinmediated capture in 24×16 rows of a microtiter plate.

FIG. 20 illustrates the analysis of DNA by distribution of 24 samples,which have initially been diluted and distributed across 24 tubes formultiplexed PCR followed by LDR with tagged probes, into 24×16 rows of amicrotiter plate.

FIG. 21 illustrates the streptavidin mediated capture of thebiotinylated PCR or RT-PCR amplicons in the wells of a microtiter platefrom FIG. 20.

FIG. 22 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 21.

FIG. 23 illustrates a hypothetic signal pattern generated from thereal-time detection of LDR-FRET in each well on a microtiter plate fromFIG. 22.

FIG. 24 illustrates the workflow for the analysis of mRNA & miRNA bydistribution of one sample, which has initially been diluted across 24tubes for multiplexed PCR followed by LDR with tagged probes, forstreptavidin mediated capture in 24×16 rows of a microtiter plate.

FIG. 25 illustrates the analysis of mRNA or miRNA, by distribution ofone sample, which has initially been serially diluted into 24 tubes formultiplexed reverse-transcriptase PCR followed by LDR with taggedprobes, into 24×16 rows of a microtiter plate.

FIG. 26 illustrates the streptavidin mediated capture of RT-PCRamplicons in the wells of a microtiter plate from FIG. 25.

FIG. 27 illustrates the addition of 16 different tag primer sets acrossthe 24 columns of a microtiter plate from FIG. 26.

FIG. 28 illustrates a hypothetic signal pattern generated from thereal-time detection of LDR-FRET in each well on a microtiter plate fromFIG. 27.

FIG. 29 illustrates a simulation of a Poisson distribution of 6 to 48molecules distributed across 24 wells. At top is a tabular form ofnumber of starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 30 illustrates a simulation of a Poisson distribution of 12 to 96molecules distributed across 24 wells. At top is a tabular form ofnumber of starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 31 illustrates a simulation of a Poisson distribution of 12 to 96molecules distributed across 48 wells. At top is a tabular form ofnumber of starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 32 illustrates a simulation of a Poisson distribution of 24 to 192molecules distributed across 48 wells. At top is a tabular form ofnumber of starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 33 illustrates a simulation of a Poisson distribution of 1 to 8molecules distributed across 8 wells. At top is a tabular form of numberof starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 34 illustrates a simulation of a Poisson distribution of 2 to 16molecules distributed across 8 wells. At top is a tabular form of numberof starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 35 illustrates a simulation of a Poisson distribution of 4 to 32molecules distributed across 8 wells. At top is a tabular form of numberof starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 36 illustrates a simulation of a Poisson distribution of 8 to 64molecules distributed across 8 wells. At top is a tabular form of numberof starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 37 illustrates a simulation of a Poisson distribution of 16 to 128molecules distributed across 8 wells. At top is a tabular form of numberof starting molecules versus molecules per well. At bottom is ahistogram of # molecules (x) versus # of wells (y).

FIG. 38 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify target(s) and/ormutations.

FIG. 39 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify target(s) and/or mutations.

FIG. 40 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify target(s) and/ormutations.

FIG. 41 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify target(s) and/ormutations.

FIG. 42 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify target(s) and/or mutations.

FIG. 43 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify target(s) and/or mutations.

FIG. 44 illustrates PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify target(s) and/ormutations.

FIG. 45 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify target methylation.

FIG. 46 illustrates PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify target methylation.

FIG. 47 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify target methylation.

FIG. 48 illustrates Nuclease-Ligation-PCR-qPCR carryover preventionreaction with Taqman detection to identify or relatively quantify targetmethylation.

FIG. 49 illustrates Nuclease-Ligation-PCR-qPCR carryover preventionreaction with UniTaq detection to identify or relatively quantify targetmethylation.

FIG. 50 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify target methylation.

FIG. 51 illustrates PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify target methylation.

FIG. 52 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify target methylation.

FIG. 53 illustrates PCR-qPCR carryover prevention reaction with Taqmandetection to identify or relatively quantify target methylation.

FIG. 54 illustrates an overview of PCR-LDR-qPCR carryover preventionreaction to identify or relatively quantify translocations at the mRNAlevel.

FIG. 55 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify translocations atthe mRNA level.

FIG. 56 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify translocations atthe mRNA level.

FIG. 57 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify translocations at the mRNAlevel.

FIG. 58 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to identify or relatively quantify alternative splicing.

FIG. 59 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify wild-type andalternatively spliced transcripts.

FIG. 60 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify wild-type andalternatively spliced transcripts.

FIG. 61 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify wild-type and alternativelyspliced transcripts.

FIG. 62 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify low-levelalternatively spliced transcripts.

FIG. 63 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify low-levelalternatively spliced transcripts.

FIG. 64 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify low-level alternativelyspliced transcripts.

FIG. 65 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to identify or relatively quantify alternative splicing.

FIG. 66 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify wild-type andalternative transcript start site.

FIG. 67 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify wild-type andalternative transcript start site.

FIG. 68 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify wild-type and alternativetranscript start site.

FIG. 69 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify low level ofalternative transcript start site.

FIG. 70 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify low level ofalternative transcript start site.

FIG. 71 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify low level of alternativetranscript start site.

FIG. 72 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to identify or relatively quantify exon deletion.

FIG. 73 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify wild-type andalternatively spliced (exon deletion) transcript.

FIG. 74 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify wild-type andalternatively spliced (exon deletion) transcript.

FIG. 75 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify wild-type and alternativelyspliced (exon deletion) transcript.

FIG. 76 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify low-levelalternatively spliced (exon deletion) transcript.

FIG. 77 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify low-levelalternatively spliced (exon deletion) transcript.

FIG. 78 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify low-level alternativelyspliced (exon deletion) transcript.

FIG. 79 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to identify or relatively quantify alternative splicing withintron insertion.

FIG. 80 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify wild-type andalternatively spliced (intron insertion) transcript.

FIG. 81 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify wild-type andalternatively spliced (intron insertion) transcript.

FIG. 82 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify wild-type and alternativelyspliced (intron insertion) transcript.

FIG. 83 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify low-levelalternatively spliced (intron insertion) transcript.

FIG. 84 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify low-levelalternatively spliced (intron insertion) transcript.

FIG. 85 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify low-level alternativelyspliced (intron insertion) transcript.

FIG. 86 illustrates PCR-LDR-qPCR carryover prevention reaction withTaqman detection to enumerate DNA copy number.

FIG. 87 illustrates PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to enumerate DNA copy number.

FIG. 88 illustrates PCR-qLDR carryover prevention reaction with FRETdetection to enumerate DNA copy number.

FIG. 89 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to enumerate RNA copy number.

FIG. 90 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to enumerate RNA copy number.

FIG. 91 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to enumerate RNA copy number.

FIG. 92 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withTaqman detection to identify or relatively quantify miRNA.

FIG. 93 illustrates RT-PCR-LDR-qPCR carryover prevention reaction withUniTaq detection to identify or relatively quantify miRNA.

FIG. 94 illustrates RT-PCR-qLDR carryover prevention reaction with FRETdetection to identify or relatively quantify miRNA.

FIG. 95 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction with Taqman detection to identify or relatively quantify miRNA.

FIG. 96 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction with UniTaq detection to identify or relatively quantify miRNA.

FIG. 97 illustrates Ligation-RT-PCR-qLDR carryover prevention reactionwith FRET detection to identify or relatively quantify miRNA.

FIG. 98 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction with Taqman detection to identify or relatively quantify miRNA.

FIG. 99 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction with UniTaq detection to identify or relatively quantify miRNA.

FIG. 100 illustrates Ligation-RT-PCR-qLDR carryover prevention reactionwith FRET detection to identify or relatively quantify miRNA.

FIG. 101 illustrates the dimensions of one version of a commerciallyavailable 384 well microtiter plate.

FIG. 102 illustrates the dimensions of another version of a commerciallyavailable 384 well microtiter plate.

FIG. 103 shows a top and side view of a typical 384 well microtiterplate configuration.

FIG. 104 shows a perspective view of a typical 384 well microtiter plateconfiguration.

FIG. 105 illustrates a top and side view of the intermediate layer of asample dispersion device positioned above several wells of a microtiterplate.

FIG. 106 illustrates an exploded perspective view of the intermediatelayer of a sample dispersion device positioned above several wells of amicrotiter plate.

FIG. 107 illustrates a top and side view of the first and intermediatelayers of a sample dispersion device positioned above several wells of amicrotiter plate.

FIG. 108 illustrates an exploded perspective view of the first andintermediate layers of a sample dispersion device positioned aboveseveral wells of a microtiter plate.

FIG. 109 illustrates a top and side view of the third, first, andintermediate layers of a sample dispersion device positioned aboveseveral wells of a microtiter plate.

FIG. 110 illustrates an exploded perspective view of the third, first,and intermediate layers of a sample dispersion device positioned aboveseveral wells of a microtiter plate.

FIG. 111 illustrates a top and side view of the second, third, first,and intermediate layers of a sample dispersion device positioned aboveseveral wells of a microtiter plate.

FIG. 112 illustrates an exploded perspective view of the second, third,first, and intermediate layers of a sample dispersion device positionedabove several wells of a microtiter plate.

FIG. 113 illustrates a top and side view of the first and intermediatelayers of a sample dispersion device that uses an alternative fillingport to pressure fill the channels and metering chambers of each row ofa microtiter plate.

FIG. 114 illustrates an exploded perspective view of the first andintermediate layers of a sample dispersion device that uses analternative filling port to pressure fill the channels and meteringchambers of each row of a microtiter plate.

FIG. 115 illustrates a top and side view of the third, first, andintermediate layers of a sample dispersion device that uses analternative filling port to pressure fill the channels and meteringchambers of each row of a microtiter plate.

FIG. 116 illustrates an exploded perspective view of the third, first,and intermediate layers of a sample dispersion device that uses analternative filling port to pressure fill the channels and meteringchambers of each row of a microtiter plate.

FIG. 117 illustrates a top and side view of the second, third, first,and intermediate layers of a sample dispersion device that uses analternative filling port to pressure fill the channels and meteringchambers of each row of a microtiter plate.

FIG. 118 illustrates an exploded perspective view of the second, third,first, and intermediate layers of a sample dispersion device that usesan alternative filling port to pressure fill the channels and meteringchambers of each row of a microtiter plate.

FIG. 119 illustrates a top and side view of the intermediate layer onthe exit side of a sample dispersion device that independently addresseseach row of a microtiter plate.

FIG. 120 illustrates an exploded perspective view of the intermediatelayer on the exit side of a sample dispersion device that independentlyaddresses each row of a microtiter plate.

FIG. 121 illustrates a top and side view of the first and intermediatelayers on the exit side of a sample dispersion device that independentlyaddresses each row of a microtiter plate.

FIG. 122 illustrates an exploded perspective view of the first andintermediate layers on the exit side of a sample dispersion device thatindependently addresses each row of a microtiter plate.

FIG. 123 illustrates a top and side view of the third, first, andintermediate layers on the exit side of a sample dispersion device thatindependently addresses each row of a microtiter plate.

FIG. 124 illustrates an exploded perspective view of the third, first,and intermediate layers on the exit side of a sample dispersion devicethat independently addresses each row of a microtiter plate.

FIG. 125 illustrates a top and side view of the second, third, first,and intermediate layers on the exit side of a sample dispersion devicethat independently addresses each row of a microtiter plate.

FIG. 126 illustrates an exploded perspective view of the second, third,first, and intermediate layers on the exit side of a sample dispersiondevice that independently addresses each row of a microtiter plate.

FIG. 127 illustrates a top and side view of the intermediate layer of asample dispersion device that independently addresses each row from bothsides of a microtiter plate.

FIG. 128 illustrates an exploded perspective view of the intermediatelayer a sample dispersion device that independently addresses each rowfrom both sides of a microtiter plate.

FIG. 129 illustrates a top and side view of the first and intermediatelayers of a sample dispersion device that independently addresses eachrow from both sides of a microtiter plate.

FIG. 130 illustrates an exploded perspective view of the first andintermediate layers of a sample dispersion device that independentlyaddresses each row from both sides of a microtiter plate.

FIG. 131 illustrates a top and side view of the third, first, andintermediate layers of a sample dispersion device that independentlyaddresses each row from both sides of a microtiter plate.

FIG. 132 illustrates an exploded perspective view of the third, first,and intermediate layers of a sample dispersion device that independentlyaddresses each row from both sides of a microtiter plate.

FIG. 133 illustrates a top and side view of the second, third, first,and intermediate layers of a sample dispersion device that independentlyaddresses each row from both sides of a microtiter plate.

FIG. 134 illustrates an exploded perspective view of the second, third,first, and intermediate layers of a sample dispersion device thatindependently addresses each row from both sides of a microtiter plate.

FIG. 135 illustrates a top and side view of the intermediate layer of asample dispersion device that independently addresses each row and eachcolumn of a microtiter plate.

FIG. 136 illustrates an exploded perspective view of the intermediatelayer of a sample dispersion device that independently addresses eachrow and each column of a microtiter plate.

FIG. 137 illustrates a top and side view of a first region of the firstlayer and the intermediate layer of a sample dispersion device thatindependently addresses each row and each column of a microtiter plate.

FIG. 138 illustrates an exploded perspective view of the first region ofthe first layer and the intermediate layer of a sample dispersion devicethat independently addresses each row and each column of a microtiterplate.

FIG. 139 illustrates a top and side view of the first and second regionsof the first layer and the intermediate layer of a sample dispersiondevice that independently addresses each row and each column of amicrotiter plate.

FIG. 140 illustrates an exploded perspective view of the first andsecond regions of the first layer and the intermediate layer of a sampledispersion device that independently addresses each row and each columnof a microtiter plate.

FIG. 141 illustrates a top and side view of the third layer, first layer(with first and second regions), and intermediate layer of a sampledispersion device that independently addresses each row and each columnof a microtiter plate.

FIG. 142 illustrates an exploded perspective view of the third layer,first layer (with first and second regions), and intermediate layer of asample dispersion device that independently addresses each row and eachcolumn of a microtiter plate.

FIG. 143 illustrates a top and side view of the second layer, thirdlayer, first layer (with first and second regions), and intermediatelayer of a sample dispersion device that independently addresses eachrow and each column of a microtiter plate.

FIG. 144 illustrates an exploded perspective view of the second layer,third layer, first layer (with first and second regions), andintermediate layer of a sample dispersion device that independentlyaddresses each row and each column of a microtiter plate.

FIG. 145 illustrates a PCR-LDR-qPCR carryover prevention reaction withTaqman readout to identify or relatively quantify low-level mutations.

FIG. 146 illustrates a PCR-LDR-qPCR carryover prevention reaction withUniTaq readout to identify or relatively quantify low-level mutations.

FIG. 147 illustrates a PCR-qLDR carryover prevention reaction with FRETreadout to identify or relatively quantify low-level mutations.

FIG. 148 illustrates a PCR-LDR-qPCR carryover prevention reaction withTaqman readout to identify or relatively quantify low-level mutations.

FIG. 149 illustrates a PCR-LDR-qPCR carryover prevention reaction withUniTaq readout to identify or relatively quantify low-level mutations.

FIG. 150 illustrates a PCR-qLDR carryover prevention reaction with FRETreadout to identify or relatively quantify low-level mutations.

FIG. 151 illustrates a PCR-LDR-qPCR carryover prevention reaction withTaqman readout to identify or relatively quantify low-level targetmethylation.

FIG. 152 illustrates a PCR-LDR-qPCR carryover prevention reaction withUniTaq readout to identify or relatively quantify low-level targetmethylation.

FIG. 153 illustrates a PCR-qLDR carryover prevention reaction with FRETreadout to identify or relatively quantify low-level target methylation.

FIG. 154 illustrates a Loop-PCR-LDR-qPCR carryover prevention reactionwith Taqman readout to identify or relatively quantify low-leveltarget(s) and/or mutations.

FIG. 155 illustrates a Loop-PCR-qLDR carryover prevention reaction withFRET readout to identify or relatively quantify low-level mutations.

FIG. 156 illustrates a Loop-PCR-LDR-qPCR carryover prevention reactionwith Taqman readout to identify or relatively quantify low-level targetmethylation.

FIG. 157 illustrates a Loop-PCR-qLDR carryover prevention reaction withFRET readout to identify or relatively quantify low-level targetmethylation.

FIG. 158 illustrates a PCR-qPCR carryover prevention reaction withTaqman readout to identify or relatively quantify low-level targetmethylation.

FIG. 159 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the BRAF V600E mutation in anexcess of wild-type DNA.

FIG. 160 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theBRAF V600E mutation.

FIG. 161 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theBRAF V600E mutation in the presence of an excess of wild-type DNA fromplasma.

FIG. 162 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the TP53 R248Q mutation in thepresence of an excess of wild-type DNA.

FIG. 163 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the TP53 R248Q mutation in thepresence of an excess of wild-type DNA.

FIG. 164 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theTP53 R248Q mutation in the presence of an excess of wild-type DNA.

FIG. 165 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theTP53 R248Q mutation in the presence of an excess of wild-type DNA fromplasma.

FIG. 166 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the KRAS G12C mutation in thepresence of an excess of wild-type DNA.

FIG. 167 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the KRAS G12S mutation in thepresence of an excess of wild-type DNA.

FIG. 168 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theKRAS G12C mutation in the presence of an excess of wild-type DNA fromplasma.

FIG. 169 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the KRAS G12D mutation in thepresence of an excess of wild-type DNA.

FIG. 170 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the KRAS G12A mutation in thepresence of an excess of wild-type DNA.

FIG. 171 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the KRAS G12V mutation in thepresence of an excess of wild-type DNA.

FIG. 172 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules of theKRAS G12V mutation in the presence of an excess of wild-type DNA inplasma.

FIG. 173 illustrates the real-time PCR amplification plots obtained inthe PCR-LDR-qPCR experiments to detect the presence or absence ofmethylation of the Vimentin gene.

FIG. 174 illustrates the real-time PCR amplification plots obtained inthe pixel PCR-LDR-qPCR experiments to enumerate single molecules ofmethylated DNA in the presence of an excess of unmethylated DNA (hgDNA).

FIG. 175 illustrates the real-time PCR amplification plots obtained inthe experiment to detect methylation of the VIM S3 top strand, using theTaqman probe version “A.”

FIG. 176 illustrates the real-time PCR amplification plots obtained inthe experiment to detect methylation of the VIM S3 bottom strand, usingthe Taqman probe version “A.”

FIG. 177 illustrates the real-time PCR amplification plots obtained inthe experiment to detect methylation of the VIM S3 top strand, using theTaqman probe version “B.”

FIG. 178 illustrates the real-time PCR amplification plots obtained inthe experiment to detect methylation of the VIM S3 bottom strand, usingthe Taqman probe version “B.”

DETAILED DESCRIPTION OF THE INVENTION

A Universal Design for Early Detection of Disease Using “Disease MarkerLoad”

The most cost-effective early disease detection test may combine aninitial multiplexed coupled amplification and ligation assay todetermine “disease load”. For cancer detection this would achieve >95%sensitivity for all cancers (pan-oncology), at >97% specificity. A flowchart for a cancer tumor load assay is illustrated in FIG. 1. An initialmultiplexed PCR/LDR screening assay scoring for mutation, methylation,miRNA, mRNA, alternative splicing, and translocations identifies thosesamples with >5 of 24-48 markers positive. Presumptive positive samplesare then assayed using “pixel” PCR/LDR with additional tissue-specificmarkers to validate the initial result, and identify tissue of origin.The physician may then order targeted sequencing to further guidetreatment decisions for the patient.

The present invention is directed to a universal diagnostic approachthat seeks to combine the best features of digital polymerase chainreaction (PCR), ligation detection reaction (LDR), and quantitativedetection of multiple disease markers, e.g., cancer markers. The familyof assay architecture and devices comprises three modules for PCR-LDRquantification of low-abundance disease markers from blood. Each modulemay be optimized independently, and may be manual, semi-automated, orfully automated. The design enables integrating the modules together,such that any module may be optimized independently to bring improvedperformance to the entire assay.

The first family of assay designs is based on an initial multiplexed PCRor RT-PCR amplification followed by multiplexed LDR using LDR probeshaving unique sequence tags containing primer-specific portions. Theproducts are distributed and mixed with tag primer sets withtarget-directed TaqMan™ probes, or alternatively with UniTaq primersets, and the input target nucleic acids quantified using real-time PCR

The first module takes an input sample of blood, and separates plasmafrom red blood cells (RBCs) and white blood cells (WBCs). It separatesplasma again to remove any residual cells. In addition, cell fractionscontaining all WBCs and circulating tumor cells (CTCs) and some RBCs areseparated. Exosomes are separated or affinity captured from plasma. Themodule purifies (i) RNA from the WBC and CTC fraction, (ii) miRNA andRNA from exosomes, and (iii) cell free DNA (cfDNA) from plasma.

The second module enables distribution of the above components into 24or 48 chambers or wells for spatial multiplexing with proportionalmultiplexed PCR or RT-PCR amplification of targeted gene, promoter,miRNA, or mRNA regions. These include: (i) specific splice-variant orgene-fusion mRNAs in the CTC containing WBC fraction, (ii) specificmiRNAs from exosomes, (iii) specific mRNAs from exosomes, (iv) specificcancer gene DNA regions from cfDNA, and (v) specific (methylated)promoter regions from cfDNA.

The third module enables spatial distribution of the above products intowells of a microtiter plate, e.g., in a 24×16 or 48×32 configuration.This module enables detection and enumeration of LDR products usingreal-time PCR to provide quantitative results for each disease marker.

The first and second modules may be configured to process multiplesamples simultaneously for the screening assay mode, where the LDRproducts containing sequence tags provide relative quantitative resultsusing real-time PCR readout (see FIG. 2). In this configuration, DNA andRNA isolated from various blood fractions from 24 individual samples aresubjected to multiplexed PCR-LDR and RT-PCR-LDR, then distributed down acolumn, e.g., 16 wells in the microtiter plate as illustrated in FIG. 3.Tag primer sets with target-directed TaqMan™ probes, or alternativelywith UniTaq primer sets are added across the rows as shown in FIG. 4,allowing for real-time PCR (FIG. 5). In this illustration, samples #2 &#15 have strong signal at >5 positions, so are considered potentiallypositive (pending additional verification as described more below),while sample #8 with 4 weak signals should also get additional workup.

The two modules may also be configured to process a single sample, withspatial multiplexing enabling “pixel” PCR/LDR, where the LDR productsenable enumeration of the original target molecules. This is analogousto digital PCR, but at a higher level of multiplexing (see FIG. 6). Inthis configuration the DNA and RNA from a single sample are distributedinto 24 chambers prior to multiplex PCR-LDR as shown in FIG. 7. In thisembodiment, some chambers have one or no target molecules. Aftermultiplexing, the LDR products are distributed down a column, e.g., 16wells in the microtiter plate (FIG. 7). Tag primer sets withtarget-directed TaqMan™ probes, or alternatively UniTaq primer sets, areadded across the rows (see FIG. 8), allowing for real-time PCR (see FIG.9). The results are interpreted based on Poisson distribution of Ctvalue as representing an integral multiple of a single molecule in theoriginal mix, i.e., 0, 1, 2 etc. FIGS. 29 and 30 show a Poissondistribution of 6 to 48 and 12 to 96 molecules in 24 wells,respectively, and FIGS. 31 and 32 show a Poisson distribution of 12 to96 and 24 to 192 molecules in 48 wells, respectively. FIG. 9, row Ashows (# addresses: # initial target molecule) of (17:0; 6:1; 1:2),which corresponds to 8 molecules. FIG. 9, row K shows (7:0; 10:1; 5:2;2:4), which corresponds to about 30 molecules.

A different form of dilution and distribution may be used to enumeratemolecules over a wider range, as illustrated for miRNA or mRNAquantification in FIGS. 10-13. Here the initial sample is distributedinto 8 chambers, diluted 10-fold and distributed into another 8chambers, etc. (see FIG. 11). The samples are subjected to multiplexedRT-PCR and LDR, and the LDR products are individually distributed down acolumn (FIG. 11). Tag primer sets with target-directed TaqMan™ probes,or alternatively UniTaq primer sets, are added across the rows (see FIG.12), allowing for real-time PCR and detection (see FIG. 13). For theexample of 24 chambers, this can quantify across 3 orders of magnitude,but across 48 chambers it can cover 6 orders of magnitude differences.FIGS. 33-37 show Poisson distributions of 1 to 128 molecules in 8 wells.In the example illustrated in FIG. 13, row G, the first two dilutionsgive higher signal than the last 8 wells (1:0; 3:1; 3:2; 1:4), whichcorresponds to about 14-16×100×1.25=1,750 to 2,000 molecules. Incontrast, row N of FIG. 13 gave signal only in the first 8 welldistribution (4:0; 3:1; 1:2), which corresponds to about 5-6×1.25=6 to 8molecules.

The second family of assay designs is based on an initial multiplexedPCR or RT-PCR amplification followed by distribution and capture of PCRamplified targets on the wells of a microtiter plate. A single cycle ofLDR enables capture of LDR products on the correct targets on the solidsupport, while mis-ligations are washed away. The LDR products arequantified, either through LDR-FRET, real-time PCR, or other reportersystems.

The first module takes an input sample of blood, and separates CTCs ifpresent, separates plasma from blood cells, and exosomes from plasma,and then purifies (i) DNA and RNA from CTCs if present, (ii) miRNA andRNA from exosomes, and (iii) cfDNA from plasma.

The second module enables distribution of the above components into 24or 48 chambers or wells for spatial multiplexing with proportionalmultiplexed PCR or RT-PCR amplification of targeted gene, promoter,miRNA, or mRNA regions. These include: (i) specific chromosomal regionsfor copy number enumeration from CTC's, (ii) specific splice-variant orgene-fusion mRNAs from CTC's, (iii) specific miRNAs from exosomes, (iv)specific mRNAs from exosomes, (v) specific cancer gene DNA regions fromcfDNA, and (vi) specific (methylated) promoter regions from cfDNA.

The third module enables spatial distribution of the above products downa column, e.g., 16 wells in the microtiter plate, followed by capture ofthe amplified target on the solid support, e.g., in a 24×16 or 48×32configuration. This module enables capture of LDR products on the solidsupport, followed by detection and enumeration of LDR products toprovide quantitative results for each marker.

The first and second modules may be configured to process multiplesamples simultaneously for the screening assay mode, wherein the LDRproducts provide relative quantitative results, analogous to real-timePCR readout (see schematic overview of FIG. 14). In this configuration,DNA and RNA isolated from various blood fractions from 24 individualsamples are subjected to multiplexed PCR and RT-PCR, then distributeddown a column, e.g., 16 wells in the microtiter plate, and captured onthe solid support (see FIGS. 15 and 16). LDR probes are added across therows, and ligation on correct target captures the product whileunreacted primer is washed away (FIG. 17). The LDR-FRET resultsillustrated in FIG. 18 are shown in their respective wells, although inthis module, the products may also be selectively denatured andenumerated using capillary electrophoresis to provide quantitativeresults. In this illustration (FIG. 18), samples #2 & 15 have strongsignal at >5 positions, so are presumptive positive, while sample 8 with4 weak signals should also get additional workup

The two modules may also be configured to process a single sample, withspatial multiplexing enabling “pixel” PCR/LDR, wherein the LDR productsenable enumeration of the original target molecules, analogous todigital PCR, but at a higher level of multiplexing (FIG. 19). In thisconfiguration the DNA and RNA from a single sample are distributed into24 chambers prior to multiplex PCR (FIG. 20), such that some chambershave one or no target molecules. The amplicons are distributed down acolumn, e.g., 16 wells in the microtiter plate, and captured on thesolid support (FIGS. 20 and 21). LDR probe addition and LDR productdetection is as above (FIGS. 22 and 23). The results are interpretedbased on Poisson distribution of LDR value as representing an integralmultiple of a single molecule in the original mix, i.e., 0, 1, 2 etc.FIGS. 29 and 30 show the Poisson distribution of 6-48 and 12-96molecules in 24 wells, respectively, and FIGS. 31 and 32 show thePoisson distribution of 12-96 and 24-192 molecules in 48 wells,respectively. FIG. 23, row A shows (# addresses: # initial targetmolecule) of (17:0; 6:1; 1:2), which corresponds to 8 molecules. FIG.23, row K shows (7:0; 10:1; 5:2; 2:4), which corresponds to about 30molecules.

A different form of dilution and distribution may be used to enumeratemolecules over a wider range, as illustrated for miRNA or mRNAquantification in FIGS. 24-28. Here the initial sample is distributedinto 8 chambers, diluted 10-fold and distributed into another 8chambers, etc. (FIG. 25). The samples are subjected to multiplex RT-PCRand distributed down a column. The RT-PCR products are captured on asolid support (FIG. 26) and LDR primer sets are added across the rows(see FIG. 27). For the example of 24 chambers, this can quantify across3 orders of magnitude, but across 48 chambers it can cover 6 orders ofmagnitude differences (see Poisson distributions of FIGS. 33-37). In theexample illustrated in FIG. 28, row G, the first two dilutions givehigher signal than the last 8 wells (1:0; 3:1; 3:2; 1:4), whichcorresponds to about 14-16×100×1.25=1,750 to 2,000 molecules. Incontrast, FIG. 28, row N gave signal only in the first 8 welldistribution (4:0; 3:1; 1:2), which corresponds to about 5-6×1.25=6 to 8molecules.

False-Positives and Carryover Protection

There is a technical challenge of distinguishing true signal generatedfrom the desired disease-specific nucleic acid differences vs. falsesignal generated from normal nucleic acids present in the sample vs.false signal generated in the absence of the disease-specific nucleicacid differences (i.e. somatic mutations).

A number of solutions to these challenges are presented below, but theyshare some common themes.

The first theme is multiplexing. PCR works best when primerconcentration is relatively high, from 50 nM to 500 nM, limitingmultiplexing. Further, the more PCR primer pairs added, the chances ofamplifying incorrect products or creating primer-dimers increaseexponentially. In contrast, for LDR probes, low concentrations on theorder of 4 nM to 20 nM are used, and probe-dimers are limited by therequirement for adjacent hybridization on the target to allow for aligation event. Use of low concentrations of gene-specific PCR primersor LDR probes containing universal primer sequence “tails” allows forsubsequent addition of higher concentrations of universal primers toachieve proportional amplification of the initial PCR or LDR products.Another way to avoid or minimize false PCR amplicons or primer dimers isto use PCR primers containing a few extra bases and a blocking group,which is liberated to form a free 3′OH by cleavage with a nuclease onlywhen hybridized to the target, e.g., a ribonucleotide base as theblocking group and RNase H2 as the cleaving nuclease.

The second theme is fluctuations in signal due to low input targetnucleic acids. Often, the target nucleic acid originated from a fewcells, either captured as CTCs, or from tumor cells that underwentapoptosis and released their DNA as small fragments (140-160 bp) in theserum. Under such conditions, it is preferable to perform some level ofproportional amplification to avoid missing the signal altogether orreporting inaccurate copy number due to fluctuations when distributingsmall numbers of starting molecules into individual wells (forreal-time, or droplet PCR quantification). As long as these initialuniversal amplifications are kept at a reasonable level (approximately12 to 20 cycles), the risk of carryover contamination during opening ofthe tube and distributing amplicons for subsequentdetection/quantification (using real-time, or droplet PCR) is minimized.

The third theme is target-independent signal, also known as “No TemplateControl” (NTC). This arises from either polymerase or ligase reactionsthat occur in the absence of the correct target. Some of this signal maybe minimized by judicious primer design. For ligation reactions, the5′→3′ nuclease activity of polymerase may be used to liberate the 5′phosphate of the downstream ligation primer (only when hybridized to thetarget), so it is suitable for ligation. Further specificity fordistinguishing presence of a low-level mutation may be achieved by: (i)using upstream LDR probes containing a mismatch in the 2^(nd) or 3^(rd)position from the 3′OH, (ii) using LDR probes to wild-type sequence that(optionally) ligate but do not undergo additional amplification, and(iii) using upstream LDR probes containing a few extra bases and ablocking group, which is liberated to form a free 3′OH by cleavage witha nuclease only when hybridized to the complementary target (e.g., RNaseH2 and a ribonucleotide base).

The fourth theme is either suppressed (reduced) amplification orincorrect (false) amplification due to unused primers in the reaction.One approach to eliminate such unused primers is to capture genomic ortarget or amplified target DNA on a solid support, allow ligation probesto hybridize and ligate, and then remove probes or products that are nothybridized. Alternative solutions include pre-amplification, followed bysubsequent nested LDR and/or PCR steps, such that there is a secondlevel of selection in the process.

The fifth theme is carryover prevention. Carryover signal may beeliminated by standard uracil incorporation during the universalamplification step, and using UDG (and optionally AP endonuclease) inthe pre-amplification workup procedure. Incorporation of carryoverprevention is central to the methods of the present invention asdescribed in more detail below. The initial PCR amplification isperformed using incorporation of uracil. The LDR reaction is performedwith LDR probes lacking uracil. Thus, when the LDR products aresubjected to real-time PCR quantification, addition of UDG destroys theinitial PCR products, but not the LDR products. Further, since LDR is alinear process and the tag primers use sequences absent from the humangenome, accidental carryover of LDR products back to the original PCRwill not cause template-independent amplification. Additional schemes toprovide carryover prevention with methylated targets include use ofrestriction endonucleases before amplification, or after bisulfitetreatment if using the latter approach as described infra.

Methods of Identifying Disease Markers

A first aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by one ormore nucleotides, one or more copy numbers, one or more transcriptsequences, and/or one or more methylated residues. This method involvesproviding a sample potentially containing one or more nucleic acidmolecules containing the target nucleotide sequence differing from thenucleotide sequences in other nucleic acid molecules by one or morenucleotides, one or more copy numbers, one or more transcript sequences,and/or one or more methylated residues, and contacting the sample withone or more enzymes capable of digesting deoxyuracil (dU) containingnucleic acid molecules present in the sample. One or more primaryoligonucleotide primer sets are provided, each primary oligonucleotideprimer set comprising (a) a first primary oligonucleotide primer thatcomprises a nucleotide sequence that is complementary to a sequenceadjacent to the target nucleotide sequence, and (b) a second primaryoligonucleotide primer that comprises a nucleotide sequence that iscomplementary to a portion of an extension product formed from the firstprimary oligonucleotide primer. The contacted sample is blended with theone or more primary oligonucleotide primer sets, a deoxynucleotide mixincluding dUTP, and a DNA polymerase to form a polymerase chain reactionmixture, and the polymerase chain reaction mixture is subjected to oneor more polymerase chain reaction cycles comprising a denaturationtreatment, a hybridization treatment, and an extension treatment,thereby forming primary extension products comprising the targetnucleotide sequence or a complement thereof. The method further involvesblending the primary extension products with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture. Eacholigonucleotide probe set comprises (a) a first oligonucleotide probehaving a target nucleotide sequence-specific portion, and (b) a secondoligonucleotide probe having a target nucleotide sequence-specificportion, wherein the first and second oligonucleotide probes of a probeset are configured to hybridize, in a base specific manner, adjacent toone another on a complementary target nucleotide sequence of a primaryextension product with a junction between them. The first and secondoligonucleotide probes of the one or more oligonucleotide probe sets areligated together to form ligated product sequences in the ligationreaction mixture, and the ligated product sequences in the sample aredetected and distinguished to identify the presence of one or morenucleic acid molecules containing target nucleotide sequences differingfrom nucleotide sequences in other nucleic acid molecules in the sampleby one or more nucleotides, one or more copy numbers, one or moretranscript sequences, and/or one or more methylated residues.

FIGS. 38-44 illustrate various embodiments of this aspect of the presentinvention.

FIG. 38 (steps A-F) illustrates an exemplary PCR-LDR-qPCR carryoverprevention reaction to detect mutations from genomic or cfDNA. Thismethod starts by isolating genomic DNA or cell free DNA (cfDNA) as shownin step A. As shown in FIG. 38 (step B), the DNA sample is treated withan enzyme capable of digesting deoxyuracil (dU) containing nucleic acidmolecules that may be present in the sample. Suitable enzymes include,without limitation, E. coli uracil DNA glycosylase (UDG), AntarcticThermolabile UDG, or Human single-strand-selective monofunctionaluracil-DNA Glycosylase (hSMUG1). The sample is then subject to anamplification reaction, e.g., a polymerase chain reaction (PCR) toamplify mutation containing regions of interest. The amplificationreaction is carried out using locus specific primers and adeoxynucleotide mix that includes dUTP. In one embodiment, limited cycleamplification (12-20 cycles) is performed to maintain relative ratios ofdifferent amplicons being produced. In another embodiment, the regionsof interest are amplified using 20-40 cycles. The amplified productscontain dU as shown in FIG. 38, step C, which allows for subsequenttreatment with UDG or a similar enzyme for carryover prevention.

As shown in FIG. 38 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. The upstream oligonucleotide probecontains a 5′ primer-specific portion (Ai) and the downstreamoligonucleotide probe contains a 3′ primer-specific portion (Ci′) thatpermits subsequent amplification of the ligation product. Followingligation, the ligation products are aliquot into separate wellscontaining one or more tag-specific primer pairs, each pair comprisingof matched primers Ai and Ci, treated with UDG or similar enzyme toremove dU containing amplification products or contaminants, PCRamplified, and detected. As shown in FIG. 38, steps E & F, detection ofthe ligation product can be carried out using traditional TaqMan™detection assay (see U.S. Pat. No. 6,270,967 to Whitcombe et al., andU.S. Pat. No. 7,601,821 to Anderson et al., which are herebyincorporated by reference in their entirety). For detection usingTaqMan™ an oligonucleotide probe spanning the ligation junction is usedin conjunction with primers suitable for hybridization on theprimer-specific portions of the ligation products for amplification anddetection. The TaqMan™ probe contains a fluorescent reporter group onone end (F1) and a quencher molecule (Q) on the other end that are inclose enough proximity to each other in the intact probe that thequencher molecule quenches fluorescence of the reporter group. Duringamplification, the TaqMan™ probe and upstream primer hybridize to theircomplementary regions of the ligation product. The 5′→3′ nucleaseactivity of the polymerase extends the hybridized primer and liberatesthe fluorescent group of the TaqMan™ probe to generate a detectablesignal (FIG. 38, step F). Use of dUTP during the amplification reactiongenerates products containing dU, which can subsequently be destroyedusing UDG for carryover prevention.

FIG. 39 illustrates an exemplary PCR-qLDR carryover prevention reactionto detect mutations from genomic or cfDNA. This method starts byisolating genomic DNA or cell free DNA (cfDNA) as shown in step A. Asshown in FIG. 39, step B, the DNA sample is treated with a deoxyuracil(dU) digesting enzyme, such as UDG, to digest dU containing nucleic acidmolecules that may be present in the sample, and then subject to anamplification reaction, e.g., a polymerase chain reaction (PCR) toamplify mutation containing regions of interest. The amplificationreaction is carried out using locus specific primers and adeoxynucleotide mix that includes dUTP. In one embodiment, limited cycleamplification (12-20 cycles) is performed to maintain relative ratios ofdifferent amplicons being produced. In another embodiment, the regionsof interest are amplified using 20-40 cycles. In this embodiment, thelocus specific primers also contain 5′ primer regions, e.g., universalprimer regions, which enables a subsequent universal PCR amplificationusing biotin labeled primers to append a 5′ biotin to the amplificationproducts containing the region of interest (FIG. 39, step B).

As shown in FIG. 39, step C, the amplification products incorporate dU,allowing for carryover prevention, and are captured on a solid supportvia the appended 5′ biotin moiety. The mutation of interest is detectedusing mutation specific ligation probes as illustrated in FIG. 39, stepD. In this embodiment, the first ligation probe contains a 3′ targetspecific region and a 5′ tail sequence with a donor or acceptor moietyand the second ligation probe in a probe set contains a 5′ targetspecific region and 3′ tail sequence with an acceptor or donor moiety,respectively. The 5′ and 3′ tail sequences of the ligation probes in aprobe set are complementary to each other and the acceptor and donorgroups are capable of generating a detectable signal via Försterresonance energy transfer (FRET) when brought in close proximity to eachother. Following ligation, unligated oligonucleotide probes are washedaway, and the ligation product is denatured from the immobilizedamplification products. Upon denaturation (FIG. 39, step E), thecomplementary 5′ and 3′ tail sequences of the ligation productshybridize to each other bringing the donor and acceptor groups in closeproximity to each other to generate a detectable FRET signal.

The ligation products formed in accordance with this aspect of thepresent invention can be distinguished using an alternative to FRETdetection. For example, the upstream probe may contain a fluorescentreporter group on the 5′ end followed by the tail sequence portion, aquenching group (e.g., ZEN), and the target-specific portion as shown inFIG. 39, step F. In the single-stranded form, the fluorescent group isquenched by the Zen group. Upon ligation of the upstream and downstreamligation probes and denaturation of the resulting the ligation product,the complementary 5′ and 3′ tail portions of the ligation producthybridize to form a short double stranded portion. In this formation thereporter group is no longer quenched by the quenching group and adetectable signal is produced.

FIG. 39 illustrates a biotinylated universal primer-streptavidin coatedsurface approach for capturing extension products on a solid support.Such capture may occur prior to or subsequent to the ligation step.Other approaches for linking the product to the solid support includecovalently attaching a portion of or the majority of the universalprimer to the solid support prior to PCR amplification.

In addition to capture of polymerase extension products usingbiotin-streptavidin, the primers can be designed to include a capturesequence on the 5′ end, a polymerase extension blocking group, and auniversal or target-specific portion on the 3′ end. After amplification,the 5′ capture sequence portion of the products will be single stranded,and if it is a long and/or GC rich sequence, it may be captured on acomplementary sequence under conditions which allow for denaturation ofthe non-captured strand, or removal by cleavage, e.g., lambdaexonuclease cleavage. The capture step may be enhanced by use of PNA,LNA, or other nucleotide analogues within the primer, capture probesequence or both.

In another embodiment, the primer may be covalently attached to thesolid surface using Dibenzocyclooctyl (DBCO) for copper-free clickchemistry (to an azide); 5-Octadiynyl dU for click chemistry (to anazide); Amino Modifier C6 dT (for peptide linkage); or Azide, for clickchemistry to an alkene or DBCO.

FIG. 40 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect mutations. Genomic or cfDNA is isolated (FIG. 40,step A), and the isolated DNA sample is treated with UDG to digest dUcontaining nucleic acid molecules that may be present in the sample. Inthis embodiment, initial amplification is carried out using locusspecific PCR primers that contain a cleavable blocking group at their 3′ends. The blocking group prevents non-target specific polymeraseextension and amplification. As shown in FIG. 40, step B, a suitableblocking group is an RNA base (r) that is cleaved by RNase-H (starsymbol) only upon hybridization of the primer to its complementarysequence (see e.g., Dobosy et. al. “RNase H-Dependent PCR (rhPCR):Improved Specificity and Single Nucleotide Polymorphism Detection UsingBlocked Cleavable Primers,” BMC Biotechnology 11(80): 1011 (2011), whichis hereby incorporated by reference in its entirety). Cleavage of theRNA base liberates a 3′OH suitable for extension by polymerase.

Following cleavage of the primer blocking groups, the region of interestis amplified and the PCR product contains dU allowing for carryoverprevention (FIG. 40, step C). Target-specific oligonucleotide probescontaining primer tags (Ai and Ci′) are then hybridized to the amplifiedproducts in a base specific manner, and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence (FIG. 40, step D). The ligation productsare detected using pairs of matched primers Ai and Ci, and TaqMan™probes that span the ligation junction as described in FIG. 38 (see FIG.40, steps E-F).

FIG. 41 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect mutations. Genomic or cfDNA is isolated (FIG. 41,step A), and the isolated DNA sample is treated with UDG to digest dUcontaining nucleic acid molecules that may be present in the sample(FIG. 41, step B). The region of interest is amplified using locusspecific primers and a deoxynucleotide mix that includes dUTP. In oneembodiment, limited cycle amplification (12-20 cycles) is performed tomaintain relative ratios of different amplicons being produced. Inanother embodiment, the regions of interest are amplified using 20-40cycles. The amplified products contain dU as shown in FIG. 41, step C,which allows for subsequent treatment with UDG or a similar enzyme forcarryover prevention.

As shown in FIG. 41 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product, while theupstream oligonucleotide probe having a sequence specific for detectingthe wildtype (non-mutated) nucleic acid sequence does not contain a 5′primer-specific portion. The downstream oligonucleotide probe, having asequence common to both mutant and wildtype sequences contains a 3′primer-specific portion (Ci′) that, together with the 5′ primer specificportion (Ai) of the upstream probe having a sequence specific fordetecting the mutation, permit subsequent amplification and detection ofonly mutant ligation products. As illustrated in step D of this Figure,another layer of specificity can be incorporated into the method byincluding a 3′ cleavable blocking group (Blk 3′, e.g. C3 spacer), and anRNA base (r), in the upstream ligation probe. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to generate aligation competent 3′OH group (FIG. 41, step D). Following ligation, theligation products can be detected using pairs of matched primers Ai andCi, and TaqMan™ probes that span the ligation junction as described inFIG. 38 (see FIG. 41, steps E-G), or using other suitable means known inthe art.

FIG. 42 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect mutations. Genomic or cfDNA is isolated (FIG. 42,step A), and the isolated DNA sample is treated with UDG to digest dUcontaining nucleic acid molecules that may be present in the sample(FIG. 42, step B). The region of interest is amplified using locusspecific primers and a deoxynucleotide mix that includes dUTP. In thisembodiment, the locus specific primers also contain 5′ primer regions,e.g., universal primer regions. Such sequences enable a subsequentuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest(FIG. 42, step B). The biotinylated PCR products are immobilized to asolid support and the mutation of interest is detected using mutationspecific ligation probes as illustrated in FIG. 42, step D. In thisembodiment, the ligation probes of a ligation pair capable of detectingthe mutant nucleic acid sequence (but not the wild-type sequence)contain complementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.As illustrated in step D of this Figure, another layer of specificitycan be incorporated into the method by including a 3′ cleavable blockinggroup, (e.g. C3-spacer), and an RNA base (r), in the upstream ligationprobe. Upon target-specific hybridization, RNase H (star symbol) removesthe RNA base to generate a ligation competent 3′OH group (FIG. 42, stepD). Following ligation (FIG. 42, step E), the complementary 5′ and 3′tail ends of the ligation products hybridize to each other bringingtheir respective donor and acceptor moieties in close proximity to eachother to generate a detectable FRET signal (FIG. 42, step F).

FIG. 43 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect mutations. Genomic or cfDNA is isolated (FIG. 43,step A), and the isolated DNA sample is treated with UDG to digest dUcontaining nucleic acid molecules that may be present in the sample(FIG. 43, step B). The region of interest is amplified using locusspecific primers and a deoxynucleotide mix that includes dUTP. In thisembodiment, the locus specific primers also contain 5′ primer regions,e.g., universal primer regions. These regions enable a subsequentuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest(FIG. 43, step B). The biotinylated PCR products are immobilized to asolid support and the mutation of interest is detected using mutationspecific ligation probes as illustrated in FIG. 43, step D. In thisembodiment, the oligonucleotide probes of a probe set are designed suchthat the 3′-most base of the first oligonucleotide probe is overlappedby the immediately flanking 5′-most base of the second oligonucleotideprobe that is complementary to the target nucleic acid molecule as shownin FIG. 43, step D. The overlapping nucleotide is referred to as a“flap”. When the overlapping flap nucleotide of the secondoligonucleotide probe is complementary to the target nucleic acidmolecule sequence and the same sequence as the terminating 3′ nucleotideof the first oligonucleotide probe, the phosphodiester bond immediatelyupstream of the flap nucleotide of the second oligonucleotide probe isdiscriminatingly cleaved by an enzyme having flap endonuclease (FEN) or5′ nuclease activity (e.g. the 5′-3′ exonuclease of Taq polymerase).That specific FEN activity produces a ligation competent 5′ phosphateend on the second oligonucleotide probe that is precisely positionedalongside the adjacent 3′ OH of the first oligonucleotide probe. As aconsequence of (a) target specific annealing by oligonucleotide probesadjacent to each other, (b) selective generation of 5′ phosphates onlywhen the cleaved flap nucleotide matches the template, and (c) additionof a ligase that discriminates against non-Watson-Crick pairing for the3′-base of the first oligonucleotide probe, very high target detectionspecificity and sensitivity is achieved. In accordance with thisembodiment, the oligonucleotide probes for ligation also containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.Following ligation (FIG. 43, step E), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 43, step F).

FIG. 44 illustrates another PCR-LDR-qPCR carryover prevention reactionto detect mutations. Genomic or cfDNA is isolated (FIG. 44, step A), andthe isolated DNA sample is treated with UDG to digest dU containingnucleic acid molecules that may be present in the sample (FIG. 44, stepB). The region of interest is amplified using locus specific primers anda deoxynucleotide mix that includes dUTP. In this embodiment, theligation probes are designed to contain UniTaq primer and tag sequencesto facilitate detections. The UniTaq system is fully described in U.S.Patent Application Publication No. 2011/0212846 to Spier, which ishereby incorporated by reference in its entirety. The UniTaq systeminvolves the use of three unique “tag” sequences, where at least one ofthe unique tag sequences (Ai) is present in the first oligonucleotideprobe, and the second and third unique tag portions (Bi′ and Ci′) are inthe second oligonucleotide probe sequence as shown in FIG. 44, step D.Upon ligation of oligonucleotide probes in a probe set, the resultingligation product will contain the Ai sequence—target specificsequences—Bi′ sequence—Ci′ sequence. The essence of the UniTaq approachis that both oligonucleotide probes of a ligation probe set need to becorrect in order to get a positive signal, which allows for highlymultiplexed nucleic acid detection. For example, and as describedherein, this is achieved by requiring hybridization of two parts, i.e.,two of the tags, to each other.

Prior to detecting the ligation product, the sample is treated with UDGto destroy original target amplicons allowing only authentic ligationproducts to be detected. For detection, the ligation product containingAi (a first primer-specific portion), Bi′ (a UniTaq detection portion),and Ci′ (a second primer-specific portion) is primed on both strandsusing a first oligonucleotide primer having the same nucleotide sequenceas Ai, and a second oligonucleotide primer that is complementary to Ci′(i.e., Ci). The first oligonucleotide primer also includes a UniTaqdetection probe (Bi) that has a detectable label F1 on one end and aquencher molecule (Q) on the other end (F1-Bi-Q-Ai). Optionallypositioned proximal to the quencher is a polymerase-blocking unit, e.g.,HEG, THF, Sp-18, ZEN, or any other blocker known in the art that issufficient to stop polymerase extension. PCR amplification results inthe formation of double stranded products as shown in FIG. 44, step F).In this example, a polymerase-blocking unit prevents a polymerase fromcopying the 5′ portion (Bi) of the first universal primer, such that thebottom strand of product cannot form a hairpin when it becomessingle-stranded. Formation of such a hairpin would result in the 3′ endof the stem annealing to the amplicon such that polymerase extension ofthis 3′ end would terminate the PCR reaction.

The double stranded PCR products are denatured, and when the temperatureis subsequently decreased, the upper strand of product forms a hairpinhaving a stem between the 5′ portion (Bi) of the first oligonucleotideprimer and portion Bi′ at the opposite end of the strand (FIG. 44, stepG). Also during this step, the second oligonucleotide primer anneals tothe 5′-primer specific portion (Ci′) of the hairpinned product. Uponextension of the second universal primer in step G, 5′ nuclease activityof the polymerase cleaves the detectable label D1 or the quenchermolecule from the 5′ end of the amplicon, thereby increasing thedistance between the label and the quencher and permitting detection ofthe label.

FIG. 145 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 145, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 145, step B). The region of interest is selectivelyamplified using mutation-selective upstream primers, locus-specificdownstream primers, and a deoxynucleotide mix that includes dUTP. Asillustrated in this Figure, another layer of selectivity can beincorporated into the method by including a 3′ cleavable blocking group(Blk 3′, e.g. C3 spacer), and a mutation-specific RNA base (mr), in theupstream mutation-specific primer. Upon target-specific hybridization,RNase H (star symbol) removes the RNA base to liberate a 3′OH groupsuitable for polymerase extension (FIG. 145, step B). RNaseH willpreferentially cleave the RNA base when it is perfectly matched tomutant DNA, but will be less likely to cleave the RNA base whenhybridized to wild-type DNA. Once the cleavage reaction has occurred,the polymerase faithfully extends the liberated 3′OH and copies themutant or wild-type base of the target. Thus, in contrast toallele-specific PCR, the PCR primer does not propagate a primer-derivedmutation. Instead, by copying the base through repeated cycles ofhybridization, cleavage, elongation, and denaturation, this PCRselectively amplifies mutant target over wild-type target during eachcycle of amplification. Optional primers with wild-type sequence lackthe RNA base and remain blocked, thus further reducing amplification ofwild-type sequence. Optionally aliquot sample into 24, 48, or 96 wellsprior to PCR. The amplified products contain dU as shown in FIG. 145,step C, which allows for subsequent treatment with UDG or a similarenzyme for carryover prevention.

As shown in FIG. 145 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product, while theupstream oligonucleotide probe having a sequence specific for detectingthe wild-type (non-mutated) nucleic acid sequence does not contain a 5′primer-specific portion. The downstream oligonucleotide probe, having asequence common to both mutant and wild-type sequences contains a 3′primer-specific portion (Ci′) that, together with the 5′ primer specificportion (Ai) of the upstream probe having a sequence specific fordetecting the mutation, permit subsequent amplification and detection ofonly mutant ligation products. As illustrated in step D of this Figure,another layer of specificity can be incorporated into the method byincluding a 3′ cleavable blocking group (Blk 3′, e.g. C3 spacer), and anRNA base (r), in the upstream ligation probe. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to generate aligation competent 3′OH group (FIG. 145, step D). Following ligation,the ligation products can be detected using pairs of matched primers Aiand Ci, and TaqMan™ probes that span the ligation junction as describedin FIG. 38 (see FIG. 145, steps E-G), or using other suitable meansknown in the art.

FIG. 146 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 146, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 146, step B). The region of interest is selectivelyamplified using mutation-selective upstream primers, locus-specificdownstream primers, and a deoxynucleotide mix that includes dUTP. Asillustrated in this Figure, another layer of selectivity can beincorporated into the method by including a 3′ cleavable blocking group(Blk 3′, e.g. C3 spacer), and a mutation-specific RNA base (mr), in theupstream mutation-specific primer. Upon target-specific hybridization,RNase H (star symbol) removes the RNA base to liberate a 3′OH groupsuitable for polymerase extension (FIG. 146, step B). RNaseH willpreferentially cleave the RNA base when it is perfectly matched tomutant DNA, but will be less likely to cleave the RNA base whenhybridized to wild-type DNA. Once the cleavage reaction has occurred,the polymerase faithfully extends the liberated 3′OH and copies themutant or wild-type base of the target. Thus, in contrast toallele-specific PCR, the PCR primer does not propagate a primer-derivedmutation. Instead, by copying the base through repeated cycles ofhybridization, cleavage, elongation, and denaturation, this PCRselectively amplifies mutant target over wild-type target during eachcycle of amplification. Optional primers with wild-type sequence lackthe RNA base and remain blocked, thus further reducing amplification ofwild-type sequence. Optionally aliquot sample into 12, 24, 48, or 96wells prior to PCR. The amplified products contain dU as shown in FIG.146, step C, which allows for subsequent treatment with UDG or a similarenzyme for carryover prevention.

As shown in FIG. 146 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product, while theupstream oligonucleotide probe having a sequence specific for detectingthe wild-type (non-mutated) nucleic acid sequence does not contain a 5′primer-specific portion. The downstream oligonucleotide probe, having asequence common to both mutant and wild-type sequences contains a 3′primer-specific portion (Bi′-Ci′) that, together with the 5′ primerspecific portion (Ai) of the upstream probe having a sequence specificfor detecting the mutation, permit subsequent amplification anddetection of only mutant ligation products. As illustrated in step D ofthis Figure, another layer of specificity can be incorporated into themethod by including a 3′ cleavable blocking group (Blk 3′, e.g. C3spacer), and an RNA base (r), in the upstream ligation probe. Upontarget-specific hybridization, RNase H (star symbol) removes the RNAbase to generate a ligation competent 3′OH group (FIG. 146, step D).Following ligation, the ligation products are amplified usingUniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as describedsupra for FIG. 44 (see FIG. 146, steps E-H), or using other suitablemeans known in the art.

FIG. 147 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 147, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 147, step B). The region of interest is selectivelyamplified using mutation-selective upstream primers, locus-specificdownstream primers, and a deoxynucleotide mix that includes dUTP. Asillustrated in step B of this Figure, another layer of selectivity canbe incorporated into the method by including a 3′ cleavable blockinggroup (Blk 3′, e.g. C3 spacer), and a mutation-specific RNA base (mr),in the upstream mutation-specific primer. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to liberate a3′OH group suitable for polymerase extension (FIG. 147, step B). RNaseHwill preferentially cleave the RNA base when it is perfectly matched tomutant DNA, but will be less likely to cleave the RNA base whenhybridized to wild-type DNA. Once the cleavage reaction has occurred,the polymerase faithfully extends the liberated 3′OH and copies themutant or wild-type base of the target. Thus, in contrast toallele-specific PCR, the PCR primer does not propagate a primer-derivedmutation. Instead, by copying the base through repeated cycles ofhybridization, cleavage, elongation, and denaturation, this PCRselectively amplifies mutant target over wild-type target during eachcycle of amplification. Optional primers with wild-type sequence lackthe RNA base and remain blocked, thus further reducing amplification ofwild-type sequence. In this embodiment, the downstream locus-specificprimers also contain 5′ primer regions, e.g., universal primer regions,that enables universal PCR amplification using biotin labeled primers toappend a 5′ biotin to the amplification products containing the regionof interest (FIG. 146, step B). Optionally aliquot sample into 12, 24,48, or 96 wells prior to PCR. The biotinylated PCR products areimmobilized to a solid support and the mutation of interest is detectedusing mutation specific ligation probes as illustrated in FIG. 147, stepD. In this embodiment, the ligation probes of a ligation pair capable ofdetecting the mutant nucleic acid sequence (but not the wild-typesequence) contain complementary tail sequences and an acceptor or donorgroup, respectively, capable of generating a detectable signal via FRETwhen brought in close proximity to each other as described supra forFIG. 39. As illustrated in this Figure, another layer of specificity canbe incorporated into the method by including a 3′ cleavable blockinggroup, (e.g. C3-spacer), and an RNA base (r), in the upstream ligationprobe. Upon target-specific hybridization, RNase H (star symbol) removesthe RNA base to generate a ligation competent 3′OH group (FIG. 147, stepD). Following ligation (FIG. 147, step E), the complementary 5′ and 3′tail ends of the ligation products hybridize to each other bringingtheir respective donor and acceptor moieties in close proximity to eachother to generate a detectable FRET signal (FIG. 147, step F).

FIG. 148 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 148, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 148, step B). The region of interest is selectivelyamplified using locus-specific upstream primers, locus-specificdownstream primers, a blocking LNA or PNA probe comprising wild-typesequence, and a deoxynucleotide mix that includes dUTP. In thisembodiment, another layer of selectivity can be incorporated into themethod by including a 3′ cleavable blocking group (Blk 3′, e.g. C3spacer), and an RNA base (r), in the upstream primer. Upontarget-specific hybridization, RNase H (star symbol) removes the RNAbase to liberate a 3′OH group which is a few bases upstream of themutation, and suitable for polymerase extension (FIG. 148, step B). Ablocking LNA or PNA probe comprising wild-type sequence that partiallyoverlaps with the upstream PCR primer will preferentially compete inbinding to wild-type sequence over the upstream primer, but not as muchto mutant DNA, and thus suppresses amplification of wild-type DNA duringeach round of PCR. Optionally aliquot sample into 12, 24, 48, or 96wells prior to PCR. The amplified products contain dU as shown in FIG.148, step C, which allows for subsequent treatment with UDG or a similarenzyme for carryover prevention.

As shown in FIG. 148 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product. Once again,the presence of blocking LNA or PNA probe comprising wild-type sequencesuppresses ligation to wild-type target sequence if present after theenrichment of mutant sequence during the PCR amplification step. Thedownstream oligonucleotide probe, having a sequence common to bothmutant and wild-type sequences contains a 3′ primer-specific portion(Ci′) that, together with the 5′ primer specific portion (Ai) of theupstream probe having a sequence specific for detecting the mutation,permit subsequent amplification and detection of only mutant ligationproducts. As illustrated in step D of this Figure, another layer ofspecificity can be incorporated into the method by including a 3′cleavable blocking group (Blk 3′, e.g. C3 spacer), and an RNA base (r),in the upstream ligation probe. Upon target-specific hybridization,RNase H (star symbol) removes the RNA base to generate a ligationcompetent 3′OH group (FIG. 148, step D). Following ligation, theligation products can be detected using pairs of matched primers Ai andCi, and TaqMan™ probes that span the ligation junction as describedsupra for FIG. 38 (see FIG. 148, steps E-G), or using other suitablemeans known in the art.

FIG. 149 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 149, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 149, step B). Upstream locus-specific primers are designeda few bases upstream of the mutation, and include a 3′ cleavableblocking group (Blk 3′, e.g. C3 spacer), and an RNA base (r). Upontarget-specific hybridization, RNase H (star symbol) removes the RNAbase to liberate a 3′OH that is suitable for polymerase extension (FIG.149, step B). A blocking LNA or PNA probe comprising wild-type sequencethat partially overlaps with the upstream PCR primer will preferentiallycompete in binding to wild-type sequence over the upstream primer, butnot as much to mutant DNA, and thus suppresses amplification ofwild-type DNA during each round of PCR. Optionally aliquot sample into12, 24, 48, or 96 wells prior to PCR. The amplified products contain dUas shown in FIG. 148, step C, which allows for subsequent treatment withUDG or a similar enzyme for carryover prevention.

As shown in FIG. 149 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product. Once again,the presence of blocking LNA or PNA probe comprising wild-type sequencesuppresses ligation to wild-type target sequence if present after theenrichment of mutant sequence during the PCR amplification step. Thedownstream oligonucleotide probe, having a sequence common to bothmutant and wild-type sequences contains a 3′ primer-specific portion(Bi-Ci′) that, together with the 5′ primer specific portion (Ai) of theupstream probe having a sequence specific for detecting the mutation,permit subsequent amplification and detection of only mutant ligationproducts. As illustrated in step D of this Figure, another layer ofspecificity can be incorporated into the method by including a 3′cleavable blocking group (Blk 3′, e.g. C3 spacer), and an RNA base (r),in the upstream ligation probe. Upon target-specific hybridization,RNase H (star symbol) removes the RNA base to generate a ligationcompetent 3′OH group (FIG. 149, step D). Following ligation, theligation products are amplified using UniTaq-specific primers (i.e.,F1-Bi-Q-Ai, Ci) and detected as described supra for FIG. 44 (see FIG.149, steps E-H), or using other suitable means known in the art.

FIG. 150 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect low-level mutations. Genomic or cfDNA is isolated(FIG. 150, step A), and the isolated DNA sample is treated with UDG todigest dU containing nucleic acid molecules that may be present in thesample (FIG. 150, step B). Upstream locus-specific primers are designeda few bases upstream of the mutation, and include a 3′ cleavableblocking group (Blk 3′, e.g. C3 spacer), and an RNA base (r). Upontarget-specific hybridization, RNase H (star symbol) removes the RNAbase to liberate a 3′OH that is suitable for polymerase extension (FIG.150, step B). A blocking LNA or PNA probe comprising wild-type sequencethat partially overlaps with the upstream PCR primer will preferentiallycompete in binding to wild-type sequence over the upstream primer, butnot as much to mutant DNA, and thus suppresses amplification ofwild-type DNA during each round of PCR. In this embodiment, thedownstream locus-specific primers also contain 5′ primer regions, e.g.,universal primer regions, that enables universal PCR amplification usingbiotin labeled primers to append a 5′ biotin to the amplificationproducts containing the region of interest (FIG. 150, step B).Optionally aliquot sample into 12, 24, 48, or 96 wells prior to PCR. Thebiotinylated PCR products are immobilized to a solid support and themutation of interest is detected using mutation specific ligation probesas illustrated in FIG. 150, step D. Once again, the presence of blockingLNA or PNA probe comprising wild-type sequence suppresses ligation towild-type target sequence if present after the enrichment of mutantsequence during the PCR amplification step. In this embodiment, theligation probes of a ligation pair capable of detecting the mutantnucleic acid sequence (but not the wild-type sequence) containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.As illustrated in this Figure, another layer of specificity can beincorporated into the method by including a 3′ cleavable blocking group,(e.g. C3-spacer), and an RNA base (r), in the upstream ligation probe.Upon target-specific hybridization, RNase H (star symbol) removes theRNA base to generate a ligation competent 3′OH group (FIG. 150, step D).Following ligation (FIG. 150, step E), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 150, step F).

In another embodiment of the this aspect of the present invention, thefirst primary oligonucleotide primer of the primary oligonucleotideprimer set comprises a 5′ portion having a nucleotide sequence that isthe same as a nucleotide sequence portion in a wildtype nucleic acidmolecule to which the primary oligonucleotide primer hybridizes to, buthas one or more nucleotide sequence mismatches to a correspondingnucleotide sequence portion in the target nucleic acid molecule.

In accordance with this embodiment, a polymerase lacking 5′ nuclease, 3′nuclease, and strand displacing activity is provided. Optionally, theprimary oligonucleotide primer may also contain a cleavable nucleotideor nucleotide analog which is cleaved during the hybridization step ofPCR to liberate a free 3′OH end on the oligonucleotide primer prior tosaid extension treatment. The polymerase chain reaction mixture issubjected to one or more additional polymerase chain reaction cyclescomprising a denaturation treatment wherein the extension products fromthe reaction are separated from each other, a hybridization treatmentwherein the first primary oligonucleotide primer hybridizes to theextension product arising from the second primary oligonucleotideprimer. The extension products arising from the second primaryoligonucleotide are capable of forming an intramolecular loop-hairpinbetween the 3′ end and the complementary sequence within the extensionproduct, which (i) comprises a mismatch at or near the 3′ end thatinhibits self-extension if hybridized to mutant target-sequence or (ii)comprises a match at the 3′ end that enhances self-extension ifself-hybridized to wild-type target-sequence. The second primaryoligonucleotide primer hybridizes to the extension product arising fromthe first primary oligonucleotide primer. The extension product of thefirst primary primer forms an intramolecular loop-hairpin between the 5′portion and the complementary sequence within the extension product.During the extension step of the PCR, the first primary oligonucleotideprimer (i) preferentially extends on extension product comprising mutanttarget sequence thereby preferentially forming primary extensionproducts comprising the mutant target nucleotide sequence or acomplement thereof, or (ii) is inhibited from forming primary extensionproducts comprising the wild-type target nucleotide sequence or acomplement thereof due to prior self-hybridization and self-extension onsaid target. The second primary oligonucleotide primer extends onextension product independent of target sequence, wherein the mutantsequence is preferentially amplified due to the different primaryextension products arising from the hybridization of the first primaryoligonucleotide primers to the target or copies thereof, resulting inenrichment of the mutant sequence extension product and complementsthereof during the primary polymerase chain reaction.

FIGS. 154 and 155 illustrate the above described embodiment of thisaspect of the present invention. As shown in FIG. 154, genomic or cfDNAis isolated (FIG. 154, step A), and the isolated DNA sample is treatedwith UDG to digest dU containing nucleic acid molecules that may bepresent in the sample (FIG. 154, step B). The region of interest isselectively amplified using locus-specific upstream primers thatcomprise a 5′ portion having a nucleotide sequence that is the same as anucleotide sequence portion of the wild-type nucleic acid molecule towhich the primer hybridizes to such that the extension product iscapable of forming a loop hairpin. In other words, the 5′ portion of theupstream primer contains a nucleotide sequence that is the same as asequence portion within the antisense wild-type DNA strand orcomplementary to a sequence portion within the sense wild-type DNAstrand. The amplification reaction also contains locus-specificdownstream primers and a deoxynucleotide mix that includes dUTP. Asillustrated in step B of this Figure, another layer of selectivity canbe incorporated into the method by including a 3′ cleavable blockinggroup (Blk 3′, e.g. C3 spacer), and an RNA base (r), in the upstreammutation-specific primer. Upon target-specific hybridization, RNase H(star symbol) removes the RNA base to liberate a 3′OH group suitable forpolymerase extension (FIG. 154, step B). Optionally aliquot sample into12, 24, 48, or 96 wells prior to PCR. The amplified products contain dUas shown in FIG. 154, step C, which allows for subsequent treatment withUDG or a similar enzyme for carryover prevention. The PCR is performedwith a polymerase lacking 5′ nuclease, 3′ nuclease, andstrand-displacement activity. FIG. 154, step D further illustrates thatduring subsequent rounds of PCR (i) the denatured wild-type bottomstrand forms a loop-hairpin with perfect match at the 3′ end, which isextended by polymerase, (ii) the denatured mutant bottom strand forms aloop-hairpin with at least one mismatched base at the 3′ end, whichgenerally is not extended by polymerase, (iii) the denatured top strandforms a loop-hairpin on 5′ side, which denatures during the extensionstep of PCR at 72° C. FIG. 154, step E, further illustrates that: (i)after extension of the loop-hairpin on wild-type DNA, extended hairpinsequence does not denature at 72° C. and prevents upstream primer fromgenerating full-length top strand. However, the loop-hairpin sequence ofmutant DNA (ii) does not extend on account of the 3′ mismatched base,and thus denatures at 72° C., enabling upstream primer to generatefull-length top strand. Likewise, top strand product (iii) denatures at72° C., allowing polymerase to generate full-length bottom strand. Thedifference in loop-hairpin extension preference of upstream primers withwild-type (i) and mutant (ii) template results in preferential removalof wild-type products during each cycle of amplification, and thusresults in preferential amplification of mutant DNA. The differentialextension efficiency of the 3′ end to extend the loop hairpin whenhybridized to mutant vs. wild-type DNA may be further enhanced bydesigning the 5′ portion of the upstream primer to contain a mismatch towild-type DNA in the 2^(nd) or 3^(rd) position from the end. Theextension product from the bottom primer will generate only 1 mismatchin the 2^(nd) or 3^(rd) position from the 3′ end when self-hybridizingto wild-type sequence, which will easily extend with polymerase, butwill generate 2 mismatches at the 3′ end when self-hybridizing to mutantsequence, which will not extend with polymerase.

As shown in FIG. 154 step G, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting themutation of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product, while theupstream oligonucleotide probe having a sequence specific for detectingthe wild-type (non-mutated) nucleic acid sequence does not contain a 5′primer-specific portion. The downstream oligonucleotide probe, having asequence common to both mutant and wild-type sequences contains a 3′primer-specific portion (Ci′) that, together with the 5′ primer specificportion (Ai) of the upstream probe having a sequence specific fordetecting the mutation, permits subsequent amplification and detectionof only mutant ligation products. As illustrated in step F of thisFigure, another layer of specificity can be incorporated into the methodby including a 3′ cleavable blocking group (Blk 3′, e.g. C3 spacer), andan RNA base (r), in the upstream ligation probe. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to generate aligation competent 3′OH group (FIG. 154, step H). Following ligation,the ligation products can be detected using pairs of matched primers Aiand Ci, and TaqMan™ probes that span the ligation junction as describedin FIG. 38 (see FIG. 154, steps H-J), or using other suitable meansknown in the art.

FIG. 155 illustrates another exemplary PCR-LDR carryover preventionreaction to detect mutations. Genomic or cfDNA is isolated (FIG. 155,step A), and the isolated DNA sample is treated with UDG to digest dUcontaining nucleic acid molecules that may be present in the sample(FIG. 155, step B). The region of interest is selectively amplifiedusing (i) locus-specific upstream primers that also comprise a 5′sequence portion that is complementary to wild-type sequence of the topstrand allowing for formation of loop-hairpins after extension, (ii)locus-specific downstream primers, and (iii) a deoxynucleotide mix thatincludes dUTP. As illustrated in step B of this Figure, another layer ofselectivity can be incorporated into the method by including a 3′cleavable blocking group (Blk 3′, e.g. C3 spacer), and an RNA base (r),in the upstream mutation-specific primer. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to liberate a3′OH group suitable for polymerase extension (FIG. 155, step B).Optionally aliquot sample into 12, 24, 48, or 96 wells prior to PCR. Theamplified products contain dU as shown in FIG. 155, step C, which allowsfor subsequent treatment with UDG or a similar enzyme for carryoverprevention. The PCR is performed with a polymerase lacking 5′ nuclease,3′ nuclease, and strand-displacement activity. FIG. 155 step D furtherillustrates that during subsequent rounds of PCR: (i) the denaturedwild-type bottom strand forms a loop-hairpin with perfect match at the3′ end, which is extended by polymerase, (ii) the denatured mutantbottom strand forms a loop-hairpin with at least one mismatched base atthe 3′ end, which generally is not extended by polymerase, (iii) thedenatured top strand forms a loop-hairpin on 5′ side, which denaturesduring the extension step of PCR at 72° C. FIG. 155, step E furtherillustrates that after extension of the loop-hairpin on wild-type DNA(i), extended hairpin sequence does not denature at 72° C. and preventsupstream primer from generating full-length top strand. However, theloop-hairpin sequence of mutant DNA (ii) does not extend on account ofthe 3′ mismatched base, and thus denatures at 72° C., enabling upstreamprimer to generate full-length top strand. Likewise, top strand product(iii) denatures at 72° C., allowing polymerase to generate full-lengthbottom strand. The difference in loop-hairpin extension preference ofupstream primers with wild-type (i) and mutant (ii) template results inpreferential removal of wild-type products during each cycle ofamplification, and thus results in preferential amplification of mutantDNA.

In this embodiment, the downstream locus-specific primers also contain5′ primer regions, e.g., universal primer regions, that enablesuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest(FIG. 155, step B). The biotinylated PCR products are immobilized to asolid support and the mutation of interest is detected using mutationspecific ligation probes as illustrated in FIG. 155, step G. In thisembodiment, the ligation probes of a ligation pair capable of detectingthe mutant nucleic acid sequence (but not the wild-type sequence)contain complementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.As illustrated in this Figure, another layer of specificity can beincorporated into the method by including a 3′ cleavable blocking group,(e.g. C3-spacer), and an RNA base (r), in the upstream ligation probe.Upon target-specific hybridization, RNase H (star symbol) removes theRNA base to generate a ligation competent 3′OH group (FIG. 155, step G).Following ligation (FIG. 155, step H), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 155, step I).

The ligation reaction used in the methods of the present invention iswell known in the art. Ligases suitable for ligating oligonucleotideprobes of a probe set together (optionally following cleavage of a 3′ribose and blocking group on the first oligonucleotide probe, or the 5′flap on the second oligonucleotide probe) include, without limitationTherms aquaticus ligase, E. coli ligase, T4 DNA ligase, T4 RNA ligase,Taq ligase, 9 N° ligase, and Pyrococcus ligase, or any otherthermostable ligase known in the art. In accordance with the presentinvention, the nuclease-ligation process of the present invention can becarried out by employing an oligonucleotide ligation assay (OLA)reaction (see Landegren, et al., “A Ligase-Mediated Gene DetectionTechnique,” Science 241:1077-80 (1988); Landegren, et al., “DNADiagnostics—Molecular Techniques and Automation,” Science 242:229-37(1988); and U.S. Pat. No. 4,988,617 to Landegren, et al.), a ligationdetection reaction (LDR) that utilizes one set of complementaryoligonucleotide probes (see e.g., WO 90/17239 to Barany et al, which ishereby incorporated by reference in their entirety), or a ligation chainreaction (LCR) that utilizes two sets of complementary oligonucleotideprobes see e.g., WO 90/17239 to Barany et al, which is herebyincorporated by reference in their entirety).

The oligonucleotide probes of a probe sets can be in the form ofribonucleotides, deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, peptide nucleotide analogues, modified peptidenucleotide analogues, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, and mixtures thereof.

The hybridization step in the ligase detection reaction, which ispreferably a thermal hybridization treatment, discriminates betweennucleotide sequences based on a distinguishing nucleotide at theligation junctions. The difference between the target nucleotidesequences can be, for example, a single nucleic acid base difference, anucleic acid deletion, a nucleic acid insertion, or rearrangement. Suchsequence differences involving more than one base can also be detected.Preferably, the oligonucleotide probe sets have substantially the samelength so that they hybridize to target nucleotide sequences atsubstantially similar hybridization conditions.

Ligase discrimination can be further enhanced by employing various probedesign features. For example, an intentional mismatch or nucleotideanalogue (e.g., Inosine, Nitroindole, or Nitropyrrole) can beincorporated into the first oligonucleotide probe at the 2^(nd) or3^(rd) base from the 3′ junction end to slightly destabilizehybridization of the 3′ end if it is perfectly matched at the 3′ end,but significantly destabilize hybridization of the 3′ end if it ismis-matched at the 3′ end. This design reduces inappropriatemisligations when mutant probes hybridize to wild-type target.Alternatively, RNA bases that are cleaved by RNases can be incorporatedinto the oligonucleotide probes to ensure template-dependent productformation. For example, Dobosy et. al. “RNase H-Dependent PCR (rhPCR):Improved Specificity and Single Nucleotide Polymorphism Detection UsingBlocked Cleavable Primers,” BMC Biotechnology 11(80): 1011 (2011), whichis hereby incorporated by reference in its entirety, describes using anRNA-base close to the 3′ end of an oligonucleotide probe with 3′-blockedend, and cutting it with RNase H2 generating a PCR-extendable andligatable 3′-OH. This approach can be used to generate eitherligation-competent 3′OH or 5′-P, or both, provided a ligase that canligate 5′-RNA base is utilized.

Other possible modifications included abasic sites, e.g., internalabasic furan or oxo-G. These abnormal “bases” are removed by specificenzymes to generate ligation-competent 3′-OH or 5′P sites. EndonucleaseIV, Tth EndolV (NEB) will remove abasic residues after the ligationoligonucleotides anneal to the target nucleic acid, but not from asingle-stranded DNA. Similarly, one can use oxo-G with Fpg orinosine/uracil with EndoV or Thymine glycol with EndoVIII.

Ligation discrimination can also be enhanced by using the couplednuclease-ligase reaction described in WO2013/123220 to Barany et al.,which is hereby incorporated by reference in its entirety. In thisembodiment, the first oligonucleotide probe bears a ligation competent3′ OH group while the second oligonucleotide probe bears a ligationincompetent 5′ end (i.e., an oligonucleotide probe without a 5′phosphate). The oligonucleotide probes of a probe set are designed suchthat the 3′-most base of the first oligonucleotide probe is overlappedby the immediately flanking 5′-most base of the second oligonucleotideprobe that is complementary to the target nucleic acid molecule. Theoverlapping nucleotide is referred to as a “flap”. When the overlappingflap nucleotide of the second oligonucleotide probe is complementary tothe target nucleic acid molecule sequence and the same sequence as theterminating 3′ nucleotide of the first oligonucleotide probe, thephosphodiester bond immediately upstream of the flap nucleotide of thesecond oligonucleotide probe is discriminatingly cleaved by an enzymehaving flap endonuclease (FEN) or 5′ nuclease activity. That specificFEN activity produces a novel ligation competent 5′ phosphate end on thesecond oligonucleotide probe that is precisely positioned alongside theadjacent 3′ OH of the first oligonucleotide probe to allow ligation ofthe two probes to occur. In accordance with this embodiment, flapendonucleases or 5′ nucleases that are suitable for cleaving the 5′ flapof the second oligonucleotide probe prior to ligation include, withoutlimitation, polymerases the bear 5′ nuclease activity such as E.coli DNApolymerase and polymerases from Taq and T thermophilus, as well as T4RNase H and TaqExo.

For insertions or deletions, incorporation of a matched base ornucleotide analogues (e.g., -amino-dA or 5-propynyl-dC) in the firstoligonucleotide probe at the 2^(nd) or 3^(rd) position from the junctionimproves stability and may improve discrimination of such frameshiftmutations from wild-type sequences. For insertions, use of one or morethiophosphate-modified nucleotides downstream from the desired scissilephosphate bond of the second oligonucleotide probe will preventinappropriate cleavage by the 5′ nuclease enzyme when the probes arehybridized to wild-type DNA, and thus reduce false-positive ligation onwild-type target. Likewise, for deletions, use of one or morethiophosphate-modified nucleotides upstream from the desired scissilephosphate bond of the second oligonucleotide probe will preventinappropriate cleavage by the 5′ nuclease enzyme when the probes arehybridized to wild-type DNA, and thus reduce false-positive ligation onwild-type target.

The method of the present invention can also be utilized to identify oneor more target nucleic acid sequences differing from other nucleic acidsequences in a sample by one or more methylated residues. In accordancewith this aspect of the present invention, the method further comprisescontacting the sample with at least a first methylation sensitive enzymeto form a restriction enzyme reaction mixture prior to forming apolymerase chain reaction mixture. In accordance with this aspect of thepresent invention, the first primary oligonucleotide primer comprises anucleotide sequence that is complementary to a region of the targetnucleotide sequence that is upstream of the one or more methylatedresidues and the second primary oligonucleotide primer comprises anucleotide sequence that is the same as a region of the targetnucleotide sequence that is downstream of the one or more methylatedresidues.

The first methylation sensitive enzyme cleaves nucleic acid molecules inthe sample that contain one or more unmethylated residues within atleast one methylation sensitive enzyme recognition sequence. Inaccordance with this embodiment, detecting involves detection of one ormore nucleic acid molecules containing the target nucleotide sequence,where the nucleic acid molecule originally contained one or moremethylated residues.

In accordance with this and all aspects of the present invention, a“methylation sensitive enzyme” is an endonuclease that will not cleaveor has reduced cleavage efficiency of its cognate recognition sequencein a nucleic acid molecule when the recognition sequence contains amethylated residue (i.e., it is sensitive to the presence of amethylated residue within its recognition sequence). A “methylationsensitive enzyme recognition sequence” is the cognate recognitionsequence for a methylation sensitive enzyme. In some embodiments, themethylated residue is a 5-methyl-C, within the sequence CpG (i.e.,5-methyl-CpG). A non-limiting list of methylation sensitive restrictionendonuclease enzymes that are suitable for use in the methods of thepresent invention include, without limitation, AciI, HinP1I, Hpy99I,HpyCH4IV, BstUI, HpaII, HhaI, or any combination thereof.

The method of the present invention may further comprise subjecting therestriction enzyme reaction mixture to a bisulfite treatment underconditions suitable to convert unmethylated cytosine residues to uracilresidues prior to forming a polymerase chain reaction mixture. In thisembodiment the first primary oligonucleotide primer of the primaryoligonucleotide primer set comprises a nucleotide sequence that iscomplementary to the bisulfite-treated target nucleotide sequencecontaining the one or more methylated, uncleaved restriction sites andthe second primary oligonucleotide primer of the provided primaryoligonucleotide primer set comprises a nucleotide sequence that iscomplementary to a portion of the extension product formed from thefirst oligonucleotide primer.

The method of the present invention may further involve providing one ormore second methylation sensitive enzymes that cleave nucleic acidmolecules containing unmethylated residues within a methylationsensitive enzyme recognition sequence. The at least one secondmethylation sensitive enzyme is blended with the polymerase chainreaction mixture comprising the bisulfite-treated restriction enzymereaction mixture to form a second restriction enzyme reaction mixture,where the second methylation sensitive enzyme cleaves nucleic acidmolecules potentially present in the sample that contain one or moreunmethylated residues within at least one methylation sensitive enzymerecognition sequence during said hybridization treatment.

In one embodiment of this aspect of the present invention, one or bothprimary oligonucleotide primers of the primary oligonucleotide primerset have a 3′ portion having a cleavable nucleotide or nucleotideanalogue and a blocking group, such that the 3′ end of said primer orprimers is unsuitable for polymerase extension. In accordance with thisembodiment, the method further involves cleaving the cleavablenucleotide or nucleotide analog of one or both oligonucleotide primersduring the hybridization treatment thereby liberating free 3′OH ends onone or both oligonucleotide primers prior to said extension treatment.

In one embodiment of this aspect of the present invention, the methodfurther involves providing one or more blocking oligonucleotides capableof hybridizing to a region of the bisulfite-treated target nucleotidesequence containing unmethylated residues. The polymerase chain reactionmixture comprising the bisulfite-treated restriction enzyme reactionmixture is contacted with the one or more blocking oligonucleotidesprior to subjecting the mixture to one or more polymerase chain reactioncycles. The one or more blocking oligonucleotides hybridize tocomplementary target nucleic acid sequences during said hybridizationtreatment and impede primary oligonucleotide primer extension duringsaid extension treatment.

FIGS. 45-53 illustrate various embodiments of the method of the presentinvention for detecting target nucleic acid molecules containing one ormore methylated residues.

The first step of the PCR-LDR-qPCR reaction depicted in FIG. 45 involvesthe isolation of genomic or cfDNA. Optionally, methylated DNA can beenriched using methylation specific antibodies. The sample is thentreated with methyl-sensitive restriction endonucleases, e.g., Bsh1236I(CG{circumflex over ( )}CG) and/or HinP1I (G{circumflex over ( )}CGC),and UNG (37° C., 30-60 minutes) to completely digest unmethylated DNAand prevent carryover (FIG. 45, step A). As shown in FIG. 45, step B,methylated regions of interest are amplified using PCR in presence ofdUTP using locus-specific primers. In one embodiment, limited cycleamplification (12-20 cycles) is performed to maintain relative ratios ofdifferent amplicons being produced. In another embodiment, the regionsof interest are amplified using 20-40 cycles. The PCR productsincorporate dU, allowing for carryover prevention (FIG. 45, step C), andthe products lack methyl groups, providing additional protection. Asshown in FIG. 45, step D, the methyl region-specific ligationoligonucleotide probes contain tag primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, using pairs of tag primers Aiand Ci′ and TaqMan™ probes.

Following the ligation reaction, the sample containing the ligationproduct can be aliquoted into separate wells for detection. Treatmentwith UDG destroys original target amplicons (FIG. 45, step E), allowingonly authentic LDR products to be amplified and detected. The ligationproducts are detected in this embodiment using a traditional TaqMan™detection assay (FIG. 45, steps E-F) as described supra.

FIG. 46 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect methylation. In this embodiment, genomic DNA or cfDNAis isolated and treated with methyl-sensitive restriction endonucleases,e.g., Bsh1236I (CG{circumflex over ( )}CG) and/or HinP1I (G{circumflexover ( )}CGC), and UNG (37° C., 30-60 minutes) to completely digestunmethylated DNA and prevent carryover (FIG. 46, step A). As shown inFIG. 46, step B, methylated regions of interest are amplified using PCRin presence of dUTP using locus-specific primers. In one embodiment,limited cycle amplification (12-20 cycles) is performed to maintainrelative ratios of different amplicons being produced. In anotherembodiment, the regions of interest are amplified using 20-40 cycles.Primers contain identical 8-11 base tails to prevent primer dimers. ThePCR products contain dU, allowing for carryover prevention (FIG. 46,step C).

The oligonucleotide probes utilized in this embodiment are designed tocontain the UniTaq primer and tag sequences to facilitate UniTaqdetection as described supra. Accordingly, following the ligationreaction and treatment with UDG for carryover prevention, the ligationproducts are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai,Ci) as shown in FIG. 46, steps E and F, and the amplified products aredetected as shown in FIG. 46, step G and described supra.

FIG. 47 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect methylation. In this embodiment, genomic DNA or cfDNAis isolated and treated with methyl-sensitive restriction endonucleases,e.g., Bsh1236I (CG{circumflex over ( )}CG) and/or HinP1I (G{circumflexover ( )}CGC), and UNG (37° C., 30-60 minutes) to completely digestunmethylated DNA and prevent carryover (FIG. 47, step A). As shown inFIG. 47, step B, methylated regions of interest are amplified using PCRin presence of dUTP using locus-specific primers. In this embodiment,the locus specific primers also contain 5′ primer regions, e.g.,universal primer regions, which enable a subsequent universal PCRamplification using biotin labeled primers to append a 5′ biotin to theamplification products containing the region of interest (FIG. 47, stepB). The biotinylated PCR products are immobilized to a solid support andthe mutation of interest is detected using mutation specific ligationprobes as illustrated in FIG. 47, step D. In accordance with thisembodiment, the oligonucleotide probes for ligation containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.Following ligation (FIG. 47, step D), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 47, step E).

FIG. 48 illustrates a nuclease-ligation-PCR-qPCR carryover preventionreaction to detect methylation. In this embodiment, genomic DNA or cfDNAis isolated and treated with HaeIII (GG{circumflex over ( )}CC),methyl-sensitive restriction endonucleases, e.g., Bsh1236I(CG{circumflex over ( )}CG) and/or HinP1I (G{circumflex over ( )}CGC),and UNG (37° C., 30-60 minutes) to completely digest unmethylated DNAand prevent carryover (FIG. 48, step A). As shown in FIG. 48, step B, ahairpinned oligonucleotide containing tag sequence (Ai′) is ligated tothe newly liberated phosphate of the digested target DNA in the sampletemplate strand. As illustrated in FIG. 48, step C, the sample istreated with HinP1I and Bsh1236I at 37° C. The methyl-sensitiverestriction enzymes are then heat-inactivated while activating Taqpolymerase for subsequent PCR amplification using locus-specific primerscontaining Ci tag sequence. As described above, the primers can containa cleavable blocking group, (e.g. C3-spacer), and an RNA base (r), thatis removed prior to amplification by an RNaseH2 (star symbol) treatmentonly when the primer is bound to complementary target sequence. Theunblocked primer is extended with polymerase, and the 5′ nucleaseactivity of the polymerase digests the 5′ portion of the ligated hairpinto generate a product complementary to the target containing the Ci andAi′ sequence. Unligated hair-pinned oligonucleotides extend onthemselves.

As shown in FIG. 48, step D, methyl-containing regions of interest areamplified using PCR including dUTP with tag-primers Ai and Ci. Performlimited cycle amplification (12-20) to maintain relative ratios ofdifferent amplicons. The PCR products incorporate dU, allowing forcarryover prevention (FIG. 48, step E), and the products lack methylgroups, providing additional protection. The amplified products arealiquot into separate wells for TaqMan™ detection using locus-specificprimers and TaqMan™ probe as described supra.

FIG. 49 illustrates a nuclease-ligation-PCR-qPCR carryover preventionreaction to detect methylation. PCR products containing the originallymethylated residues of interest and dU are generated following the stepsA-D as illustrated and described above with regard to FIG. 48. In thisembodiment, subsequent amplification using UniTaq primers and tagsequences are used to detect the methylated residues of interest. Asshown in step E of FIG. 49, PCR products containing dU for carryoverprevention, are aliquot into separate wells and amplified usinglocus-specific primers tailed with Aj, and Bj-Cj, as well asUniTaq-specific primers (F1-Bj-Q-Aj, and Cj). The resultingdouble-stranded DNA products are shown in FIG. 49, step F. As shown inFIG. 49, step G, after the denaturation step, the temperature is cooledto allow hairpin formation between Bj and Bj′. The 5′→3′ nucleaseactivity of Taq polymerase (filled diamonds) extends primer Ci andliberates the fluorescent group to generate signal.

FIG. 50 illustrates a PCR-LDR-qPCR carryover prevention reaction todetect methylation. In this embodiment, genomic DNA or cfDNA is isolatedand treated with methyl-sensitive restriction endonucleases, e.g.,Bsh1236I (CG{circumflex over ( )}CG) and UNG (37° C., 30-60 minutes) tocompletely digest unmethylated DNA and prevent carryover (FIG. 50, stepA). The digested DNA is bisulfite-treated to convert unmethylated dCresidues to uracil (dU) thereby rendering the double stranded DNAnon-complementary. As shown in FIG. 50B, locus-specific primers arehybridized in the presence of BstU1 (CG{circumflex over ( )}CG) (filledtriangles), which will cleave carryover DNA containing unmethylatedresidues. The locus-specific primers contain a cleavable blocker attheir 3′ end to prevent non-target specific extension. Once hybridizedto their complementary target sequence, the blocker group (C3-spacer),and an RNA base is removed with RNaseH2 (star symbol). Themethyl-containing region of interest is amplified using PCR in thepresence of dUTP. Perform limited cycle amplification (12-20) tomaintain relative ratios of different amplicons, and optionally aliquotdigested sample into 12, 24, 48, or 96 wells prior to PCR. As shown inthis embodiment, a blocking oligonucleotide (thick black bar) may beused to limit amplification of wild-type DNA.

The amplified products contain dU and lack methyl groups, allowing forcarryover prevention as shown in FIG. 50, step C. As shown in FIG. 50,step D, methyl region-specific ligation oligonucleotide probescontaining primer-specific sequences (Ai, Ci′) suitable for subsequentPCR amplification, are hybridized to the target region of interest.Ligase (filled circles) covalently seals the two oligonucleotidestogether to form ligation products containing upstream and downstreamprimer specific portions and a portion corresponding to the methylatedregion of interest. The ligation products are aliquot into separatewells for detection using pairs of matched primers Ai and Ci, andTaqMan™ probes across the ligation junction as shown in FIG. 50 stepsE-F. The sample mixture is treated with UDG for carryover prevention andremoval of original target amplicons (FIG. 50, step E) so that onlyauthentic LDR products are amplified and detected.

FIG. 51 illustrates another PCR-LDR-qPCR carryover prevention reactionto detect methylation. PCR products containing the originally methylatedresidues of interest and dU are generated following the steps A-C asillustrated and described above with regard to FIG. 50. In thisembodiment, the methyl region-specific ligation oligonucleotides containUniTaq detection primer-specific sequences (Ai and Ci′) and tag sequence(Bi′) for subsequent PCR amplification detection. The ligation productsare amplified and detected using UniTaq-specific primers (F1-Bi-Q-Ai,Ci) as described supra (FIG. 51, steps E-G).

FIG. 52 illustrates another PCR-qLDR carryover prevention reaction todetect methylation. Similar to the embodiments shown in FIGS. 50 and 51,genomic DNA or cfDNA is isolated and treated with methyl-sensitiverestriction endonucleases, e.g., Bsh1236I (CG{circumflex over ( )}CG)and UNG (37° C., 30-60 minutes) to completely digest unmethylated DNAand prevent carryover (FIG. 52, step A). The digested DNA isbisulfite-treated to convert unmethylated residues to uracil therebyrendering the double stranded DNA non-complementary. As shown in FIG.52B, locus-specific primers containing a 3′ cleavable blocking group arehybridized in the presence of BstU1 (CG{circumflex over ( )}CG) (filledtriangles), which will cleave carryover DNA containing unmethylatedresidues. Once the primers hybridize to their complementary targetsequence, the blocking group is removed and the methyl-containing regionof interest is amplified using PCR in the presence of dUTP. In thisembodiment, the locus-specific primers contain universal tails (withidentical 8-11 bases to prevent primer dimers), which enables asubsequent universal primer amplification to append a 5′ biotin group. Ablocking oligonucleotide primer may be used during amplification tolimit formation of wild-type amplicon.

Amplification products are captured on a solid support via the 5′ biotingroup (FIG. 52, step C). As shown in FIG. 52D, ligation products areformed using methyl region-specific ligation oligonucleotide probes,where the downstream probe contains a 5′ acceptor group and sequencetail that is complementary to the 3′ sequence tail of the upstreamligation probe. The upstream ligation probe also contains a 3′ donorgroup such that upon the formation of a ligation product, the 5′ and 3′complementary regions of the product hybridize bringing the acceptor anddonor groups in close proximity to each other to generate FRET signalfor detection of the methylated residues of interest (FIG. 52, step E).

FIG. 151 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level target methylation. Genomic or cfDNA isisolated (FIG. 151, step A), and the isolated DNA sample is treated withmethyl-sensitive restriction endonucleases, e.g., Bsh1236I(CG{circumflex over ( )}CG), and UNG to completely digest unmethylatedDNA and prevent carryover (FIG. 151, step A). The digested DNA isbisulfite-treated to convert unmethylated residues to uracil therebyrendering the double stranded DNA non-complementary. The region ofinterest is selectively amplified using locus-specific upstream primers,locus-specific downstream primers, a blocking LNA or PNA probecomprising the bisulfate converted unmethylated sequence or itscomplement, and a deoxynucleotide mix that includes dUTP. In thisembodiment, another layer of selectivity can be incorporated into themethod by including a 3′ cleavable blocking group (Blk 3′, e.g. C3spacer), and an RNA base (r), in the upstream primer, as well as thedownstream primer. Upon target-specific hybridization, RNase H (starsymbol) removes the RNA base to liberate a 3′OH group of the upstreamprimer, which is a few bases upstream of the methylation site, andsuitable for polymerase extension (FIG. 151, step B). A blocking LNA orPNA probe comprising the bisulfite converted unmethylated sequence (orits complement) that partially overlaps with the upstream PCR primerwill preferentially compete for binding to the bisulfite convertedunmethylated sequence over the upstream primer and over the bisulfiteconverted methylated sequence DNA, thus suppressing amplification ofbisulfite converted unmethylated sequence DNA during each round of PCR.Optionally aliquot sample into 12, 24, 48, or 96 wells prior to PCR. Theamplified products contain dU as shown in FIG. 151, step C, which allowsfor subsequent treatment with UDG or a similar enzyme for carryoverprevention.

As shown in FIG. 151 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. In this embodiment, the upstreamoligonucleotide probe having a sequence specific for detecting thebisulfite converted methylated target sequence of interest furthercontains a 5′ primer-specific portion (Ai) to facilitate subsequentdetection of the ligation product. Once again, the presence of blockingLNA or PNA probe comprising bisulfite converted unmethylated sequence(or its complement) suppresses hybridization of the upstream ligationprobe to bisulfite converted unmethylated target sequence if presentafter the enrichment of bisulfite converted methylated target sequenceduring the PCR amplification step. The downstream oligonucleotide probe,having a sequence common to both bisulfite converted unmethylated andmethylated target sequences contains a 3′ primer-specific portion (Ci′)that, together with the 5′ primer specific portion (Ai) of the upstreamprobe having a sequence specific for detecting the bisulfite convertedmethylated target sequence. Ligation of the upstream and downstreamoligonucleotide probes permits subsequent amplification and detection ofonly bisulfite converted methylated target sequence ligation products.As illustrated in step D of this Figure, another layer of specificitycan be incorporated into the method by including a 3′ cleavable blockinggroup (Blk 3′, e.g. C3 spacer), and an RNA base (r), in the upstreamligation probe. Upon target-specific hybridization, RNase H (starsymbol) removes the RNA base to generate a ligation competent 3′OH group(FIG. 151, step D). Following ligation, the ligation products can bedetected using pairs of matched primers Ai and Ci, and TaqMan™ probesthat span the ligation junction as described supra for FIG. 38 (see FIG.151, steps E-G), or using other suitable means known in the art.

FIG. 152 illustrates another exemplary PCR-LDR-qPCR carryover preventionreaction to detect low-level target methylation. Genomic or cfDNA isisolated (FIG. 152, step A), and the isolated DNA sample is treated withmethyl-sensitive restriction endonucleases, e.g., Bsh1236I (CGACG), andUNG to completely digest unmethylated DNA and prevent carryover (FIG.152, step A). The digested DNA is bisulfite-treated to convertunmethylated residues to uracil thereby rendering the double strandedDNA non-complementary. Upstream and downstream locus-specific primersare designed to include a 3′ cleavable blocking group (Blk 3′, e.g. C3spacer), and an RNA base (r). Upon target-specific hybridization, RNaseH (star symbol) removes the RNA base to liberate a 3′OH, and is suitablefor polymerase extension (FIG. 152, step B). A blocking LNA or PNA probecomprising the bisulfite converted unmethylated sequence (or itscomplement) that partially overlaps with the upstream PCR primer willpreferentially compete for binding to bisulfite converted unmethylatedtarget sequence over the upstream primer and over bisulfite convertedmethylated target sequence, thus suppressing amplification of bisulfiteconverted unmethylated target sequence during each round of PCR.Optionally aliquot sample into 12, 24, 48, or 96 wells prior to PCR. Theamplified products contain dU as shown in FIG. 152, step C, which allowsfor subsequent treatment with UDG or a similar enzyme for carryoverprevention.

As shown in FIG. 152 step D, target-specific oligonucleotide probes arehybridized to the amplified products and ligase (filled circle)covalently seals the two oligonucleotides together when hybridized totheir complementary sequence. Once again, the presence of blocking LNAor PNA probe comprising bisulfite converted unmethylated sequence (orits complement) suppresses ligation to bisulfite converted unmethylatedtarget sequence if present after the enrichment of bisulfite convertedmethylated target sequence during the PCR amplification step. In thisembodiment, the upstream oligonucleotide probe having a sequencespecific for detecting the bisulfite converted methylated targetsequence of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product. Thedownstream oligonucleotide probe, having a sequence common to bothbisulfite converted unmethylated and methylated target sequencescontains a 3′ primer-specific portion (Bi′-Ci′) that, together with the5′ primer specific portion (Ai) of the upstream probe having a sequencespecific for detecting the bisulfite converted methylated targetsequence, permit subsequent amplification and detection of onlybisulfite converted methylated target sequence ligation products. Asillustrated in step D of this Figure, another layer of specificity canbe incorporated into the method by including a 3′ cleavable blockinggroup (Blk 3′, e.g. C3 spacer), and an RNA base (r), in the upstreamligation probe. Upon target-specific hybridization, RNase H (starsymbol) removes the RNA base to generate a ligation competent 3′OH group(FIG. 152, step D). Following ligation, the ligation products areamplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) anddetected as described supra for FIG. 44 (see FIG. 152, steps E-H), orusing other suitable means known in the art.

FIG. 153 illustrates another exemplary PCR-qLDR carryover preventionreaction to detect low-level target methylation. Genomic or cfDNA isisolated (FIG. 153, step A), and the isolated DNA sample is treated withmethyl-sensitive restriction endonucleases, e.g., Bsh1236I (CGACG), andUNG to completely digest unmethylated DNA and prevent carryover (FIG.153, step A). The digested DNA is bisulfite-treated to convertunmethylated residues to uracil thereby rendering the double strandedDNA non-complementary. Upstream and downstream locus-specific primersare designed to include a 3′ cleavable blocking group (Blk 3′, e.g. C3spacer), and an RNA base (r). Upon target-specific hybridization, RNaseH (star symbol) removes the RNA base to liberate a 3′OH, and is suitablefor polymerase extension (FIG. 153, step B). A blocking LNA or PNA probecomprising the bisulfite converted unmethylated sequence (or itscomplement) that partially overlaps with the upstream PCR primer willpreferentially compete for binding to bisulfite converted unmethylatedtarget sequence over the upstream primer and over the bisulfiteconverted methylated target sequence, thus suppressing amplification ofbisulfite converted unmethylated target sequence during each round ofPCR. In this embodiment, the downstream locus-specific primers alsocontain 5′ primer regions, e.g., universal primer regions, that enablesuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest(FIG. 153, step B). Optionally aliquot sample into 12, 24, 48, or 96wells prior to PCR. The amplified products contain dU as shown in FIG.153, step C, which allows for subsequent treatment with UDG or a similarenzyme for carryover prevention. The biotinylated PCR products areimmobilized to a solid support and the bisulfite converted methylatedtarget sequence of interest is detected using bisulfite convertedmethylated target sequence specific ligation probes as illustrated inFIG. 153, step D. Once again, the presence of blocking LNA or PNA probecomprising the bisulfite converted unmethylated sequence (or itscomplement) suppresses ligation to bisulfite converted unmethylatedtarget sequence if present after the enrichment of bisulfite convertedmethylated target sequence during the PCR amplification step. In thisembodiment, the ligation probes of a ligation pair capable of detectingthe bisulfite converted methylated target sequence nucleic acid sequence(but not the bisulfite converted unmethylated target sequence) containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra for FIG. 39.As illustrated in this Figure, another layer of specificity can beincorporated into the method by including a 3′ cleavable blocking group,(e.g. C3-spacer), and an RNA base (r), in the upstream ligation probe.Upon target-specific hybridization, RNase H (star symbol) removes theRNA base to generate a ligation competent 3′OH group (FIG. 153, step D).Following ligation (FIG. 153, step E), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 153, step F).

In another embodiment of this aspect of the present invention the firstprimary oligonucleotide primer of the primary oligonucleotide primer setcomprises a 5′ portion having a nucleotide sequence that is the same asa nucleotide sequence portion in a bisulfate-treated unmethylated targetsequence to which the primary oligonucleotide primer hybridizes to, buthas one or more nucleotide sequence mismatches to a correspondingnucleotide sequence portion in the bisulfate-treated methylated targetsequence.

In accordance with this embodiment, the DNA polymerase is one that lacks5′ nuclease, 3′ nuclease, and strand displacing activity. Optionally,the primary oligonucleotide primer also contains a cleavable nucleotideor nucleotide analog that is cleaved during the hybridization step ofthe polymerase chain reaction to liberate free 3′OH ends on theoligonucleotide primer suitable for extension. The polymerase chainreaction mixture is subject to one or more additional polymerase chainreaction cycles comprising a denaturation treatment wherein theextension products from the reaction are separated from each other, anda hybridization treatment wherein the first primary oligonucleotideprimer hybridizes to the extension product arising from the secondprimary oligonucleotide primer. The extension product arising from thesecond primary primer forms an intramolecular loop-hairpin between the3′ end and the complementary sequence within the extension product,which (i) comprises one or more mismatches at or near the 3′ end thatinhibits self-extension if self-hybridized to bisulfite-treatedmethylated sequence or (ii) comprises a match at the 3′ end thatenhances self-extension if self-hybridized to bisulfate-treatedunmethylated target-sequence. The second primary oligonucleotide primerhybridizes to the extension product arising from the first primaryoligonucleotide primer. The extension product arising from the firstprimary oligonucleotide primer forms an intramolecular loop-hairpinbetween the 5′ portion and the complementary sequence within theextension product. During the extension step of the PCR, the firstprimary oligonucleotide primer (i) preferentially extends on extensionproduct comprising bisulfite-treated methylated target sequence therebypreferentially forming primary extension products comprising thebisulfite-treated methylated target nucleotide sequence or a complementthereof, or (ii) is inhibited from forming primary extension productscomprising the bisulfite-treated unmethylated target nucleotide sequenceor a complement thereof due to prior self-hybridization andself-extension on said target. The second primary oligonucleotide primer(iii) extends on extension product independent of target sequence,wherein the bisulfate-treated methylated sequence is preferentiallyamplified due to the different primary extension products arising fromthe hybridization of the first primary oligonucleotide primers to thetarget or copies thereof, resulting in enrichment of thebisulfate-treated methylated sequence extension product and complementsthereof during said the primary polymerase chain reaction

FIGS. 156 and 157 illustrate this embodiment of the present invention.As shown in FIG. 156, genomic DNA or cfDNA is isolated and treated withmethyl-sensitive restriction endonucleases, e.g., Bsh1236I (CGACG) andUNG (37° C., 30-60 minutes) to completely digest unmethylated DNA andprevent carryover (FIG. 156, step A). The digested DNA isbisulfate-treated to convert unmethylated dC residues to uracil (dU)thereby rendering the double stranded DNA non-complementary. The regionof interest is selectively amplified using (i) locus-specific upstreamprimers that also comprise a 5′ sequence portion complementary tobisulfate-treated unmethylated sequence of the top strand allowing forformation of loop-hairpins after extension, (ii) locus-specificdownstream primers, and (iii) a deoxynucleotide mix that includes dUTP.As illustrated in step B of this Figure, another layer of selectivitycan be incorporated into the method by including a 3′ cleavable blockinggroup (Blk 3′, e.g. C3 spacer), and an RNA base (r), in the upstreammutation-specific primer. Upon target-specific hybridization, RNase H(star symbol) removes the RNA base to liberate a 3′OH group suitable forpolymerase extension (FIG. 156, step B). Optionally aliquot digestedsample into 12, 24, 48, or 96 wells prior to PCR. The amplified productscontain dU as shown in FIG. 156, step C, which allows for subsequenttreatment with UDG or a similar enzyme for carryover prevention. PCR isperformed with a polymerase lacking 5′ nuclease, 3′ nuclease, andstrand-displacement activity. FIG. 156, step D further illustrates thatin subsequent rounds of amplification: (i) the denaturedbisulfate-treated unmethylated bottom strand forms a loop-hairpin withperfect match at the 3′ end, which is extended by polymerase, (ii) thedenatured bisulfate-treated methylated bottom strand forms aloop-hairpin with two or more mismatches, which generally is notextended by polymerase, and (iii) the denatured top strand forms aloop-hairpin on its 5′ side, which denatures during the extension stepof PCR at 72° C. FIG. 156, step E further illustrates that: afterextension of the loop-hairpin on bisulfate-treated unmethylated DNA (i),the extended hairpin sequence does not denature at 72° C. and preventsupstream primer from generating full-length top strand. However, theloop-hairpin sequence of bisulfate-treated methylated DNA (ii) does notextend on account of two or more mismatched bases, and thus denatures at72° C., enabling upstream primer to generate full-length top strand.Likewise, top strand product (iii) denatures at 72° C., allowingpolymerase to generate full-length bottom strand. The difference inloop-hairpin extension preference of upstream primers with (i)bisulfite-treated unmethylated and (ii) bisulfate-treated methylatedtemplate results in preferential removal of bisulfate-treatedunmethylated amplification products during each cycle of amplification,and thus results in preferential amplification of bisulfate-treatedmethylated DNA.

As shown in FIG. 156, step G, oligonucleotide probes specific for thebisulfite-treated methylated target sequence are hybridized to theamplified products, and ligase (filled circle) covalently seals the twooligonucleotides together when hybridized to their complementarysequence. In this embodiment, the upstream oligonucleotide probe havinga sequence specific for detecting the bisulfate-treated methylatedsequence of interest further contains a 5′ primer-specific portion (Ai)to facilitate subsequent detection of the ligation product, while theoptional upstream oligonucleotide probe having a sequence specific fordetecting the bisulfate-treated unmethylated nucleic acid sequence doesnot contain a 5′ primer-specific portion. The downstream oligonucleotideprobe, having a sequence specific for detecting bisulfate-treatedmethylated sequences contains a 3′ primer-specific portion (Ci′) that,together with the 5′ primer specific portion (Ai) of the upstream probehaving a sequence specific for detecting bisulfate-treated methylatedsequence of interest, permit subsequent amplification and detection ofonly mutant ligation products. As illustrated in this Figure, anotherlayer of specificity can be incorporated into the method by including a3′ cleavable blocking group (Blk 3′, e.g. C3 spacer), and an RNA base(r), in the upstream ligation probe. Upon target-specific hybridization,RNase H (star symbol) removes the RNA base to generate a ligationcompetent 3′OH group (FIG. 156, step H). Following ligation, theligation products can be detected using pairs of matched primers Ai andCi, and TaqMan™ probes that span the ligation junction as described inFIG. 38 (see FIG. 156, steps H-J), or using other suitable means knownin the art.

FIG. 157 illustrates another PCR-qLDR carryover prevention reaction todetect methylation. In this embodiment, genomic DNA or cfDNA is isolatedand treated with methyl-sensitive restriction endonucleases, e.g.,Bsh1236I (CG{circumflex over ( )}CG) and UNG (37° C., 30-60 minutes) tocompletely digest unmethylated DNA and prevent carryover (FIG. 157, stepA). The digested DNA is bisulfite-treated to convert unmethylated dCresidues to uracil (dU) thereby rendering the double stranded DNAnon-complementary. The region of interest is selectively amplified using(i) locus-specific upstream primers that also comprise a 5′ portionhaving a sequence complementary to bisulfate-treated unmethylatedsequence of the top strand allowing for formation of loop-hairpins afterextension, (ii) locus-specific downstream primers, and (iii) adeoxynucleotide mix that includes dUTP. As illustrated in this Figure,another layer of selectivity can be incorporated into the method byincluding a 3′ cleavable blocking group (Blk 3′, e.g. C3 spacer), and anRNA base (r), in the upstream primer. Upon target-specifichybridization, RNase H (star symbol) removes the RNA base to liberate a3′OH group suitable for polymerase extension (FIG. 157, step B).Optionally aliquot digested sample into 12, 24, 48, or 96 wells prior toPCR. The amplified products contain dU as shown in FIG. 157, step C,which allows for subsequent treatment with UDG or a similar enzyme forcarryover prevention. PCR is performed with a polymerase lacking 5′nuclease, 3′ nuclease, and strand-displacement activity. FIG. 157, stepD further illustrates that in subsequent rounds of amplification (i) thedenatured bisulfate-treated unmethylated bottom strand forms aloop-hairpin with perfect match at the 3′ end, which is extended bypolymerase, (ii) the denatured bisulfite-treated methylated bottomstrand forms a loop-hairpin with two or more mismatches, which generallyis not extended by polymerase, and (iii) the denatured top strand formsa loop-hairpin on 5′ side, which denatures during the extension step ofPCR at 72° C. FIG. 157, step E further illustrates that after extensionof the loop-hairpin on bisulfate-treated unmethylated DNA (i), extendedhairpin sequence does not denature at 72° C. and prevents upstreamprimer from generating full-length top strand. However, the loop-hairpinsequence of bisulfate-treated methylated DNA (ii) does not extend onaccount of two or more mismatched bases, and thus denatures at 72° C.,enabling upstream primer to generate full-length top strand. Likewise,top strand product denatures at 72° C., allowing polymerase to generatefull-length bottom strand (iii). The difference in loop-hairpinextension preference of upstream primers with (i) bisulfate-treatedunmethylated and (ii) bisulfite-treated methylated template results inpreferential removal of bisulfate-treated unmethylated amplificationproducts during each cycle of amplification, and thus results inpreferential amplification of bisulfate-treated methylated DNA.

In this embodiment, the downstream locus-specific primers also contain a5′ primer region, e.g., universal primer region, that enables universalPCR amplification using biotin labeled primers to append a 5′ biotin tothe amplification products containing the region of interest (FIG. 157,step B). The biotinylated PCR products are immobilized to a solidsupport and the bisulfate-treated methylated sequence of interest isdetected using ligation probes specific for the bisulfate-treatedmethylated target sequence as illustrated in FIG. 150, step G. In thisembodiment, the ligation probes of a ligation pair capable of detectingthe bisulfate-treated methylated nucleic acid sequence (but not thebisulfate-treated unmethylated sequence) contain complementary tailsequences and an acceptor or donor group, respectively, capable ofgenerating a detectable signal via FRET when brought in close proximityto each other as described supra for FIG. 39. As illustrated in step Gof this Figure, another layer of specificity can be incorporated intothe method by including a 3′ cleavable blocking group, (e.g. C3-spacer),and an RNA base (r), in the upstream ligation probe. Upontarget-specific hybridization, RNase H (star symbol) removes the RNAbase to generate a ligation competent 3′OH group (FIG. 150, step G).Following ligation (FIG. 157, step H), the complementary 5′ and 3′ tailends of the ligation products hybridize to each other bringing theirrespective donor and acceptor moieties in close proximity to each otherto generate a detectable FRET signal (FIG. 157, step I).

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more nucleic acid molecules containinga target nucleotide sequence differing from nucleotide sequences inother nucleic acid molecules in the sample, or other samples, by onemore methylated residue. This method involves providing a samplecontaining one or more nucleic acid molecules potentially comprising thetarget nucleotide sequence differing from the nucleotide sequences inother nucleic acid molecules by one or more methylated residues andcontacting the sample with one or more enzymes capable of digestingdeoxyuracil (dU) containing nucleic acid molecules present in thesample. The method further involves contacting the sample with one ormore methylation sensitive enzymes to form a restriction enzyme reactionmixture, wherein the one or more said methylation sensitive enzymecleaves nucleic acid molecules in the sample that contain one or moreunmethylated residues within at least one methylation sensitive enzymerecognition sequence. One or more primary oligonucleotide primer setsare provided, each primary oligonucleotide primer set comprising (a)first primary oligonucleotide primer comprising a nucleotide sequencethat is complementary to a region of the target nucleotide sequence thatis upstream of the one or more methylated residues and (b) a secondprimary oligonucleotide primer comprising a nucleotide sequence that isthe same as a region of the target nucleotide sequence that isdownstream of the one or more methylated residues. The restrictionenzyme reaction mixture is blended with the one or more primaryoligonucleotide primer sets, a deoxynucleotide mix including dUTP, and aDNA polymerase to form a primary polymerase chain reaction mixture. Themethod further involves subjecting the primary polymerase chain reactionmixture to one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment, thereby forming primary extension products comprising thetarget nucleotide sequence or a complement thereof. One or moresecondary oligonucleotide primer sets are provided, each secondaryoligonucleotide primer set comprising first and second nestedoligonucleotide primers capable of hybridizing to the primary extensionproducts The primary extension products are blended with the one or moresecondary oligonucleotide primer sets, a deoxynucleotide mix includingdUTP, and a DNA polymerase to form a secondary polymerase chain reactionmixture, and the secondary polymerase chain reaction mixture issubjected to one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming secondary extension products. The secondaryextension products in the sample are detected and distinguished toidentify the presence of one or more nucleic acid molecules containingtarget nucleotide sequences differing from nucleotide sequences in othernucleic acid molecules in the sample by one or more methylated residues.

In accordance with this aspect of the present invention, the secondarypolymerase chain reaction mixture may further comprise one or moreoligonucleotide detection probes, e.g., a TaqMan™ oligonucleotidedetection probe. The detection probe hybridizes to a target nucleotidesequence within the primary extension product or its complement, and hasa quencher molecule and a detectable label that are separated from eachother but in close enough proximity to each so that the quenchermolecule quenches the detectable label. During the hybridization step ofthe secondary polymerase chain reaction process, the one or moreoligonucleotide detection probes hybridize to complementary portions ofthe primary extension products and the quencher molecule and thedetectable label are subsequently cleaved from the one or moreoligonucleotide detection probes during the extension step. Uponcleavage, the detectable label is separated from the quencher so thatthe detectable label is detected.

In one embodiment, one or both primary oligonucleotide primers of theprimary oligonucleotide primer set may optionally have a 3′ portioncomprising a cleavable nucleotide or nucleotide analogue and a blockinggroup, such that the 3′ end of said primer or primers is unsuitable forpolymerase extension until the cleavable nucleotide or nucleotide analogis cleaved. Upon cleavage, a free 3′OH end is liberated on one or bothprimary oligonucleotide primers prior to allow for primer extension.

In another embodiment, the primary oligonucleotide primers of theprimary oligonucleotide primer set comprise an identical orsubstantially identical 5′ nucleotide sequence portion that is betweenabout 6 to 20 bases in length. In accordance with this embodiment, thedesired extension products that are generated are of sufficient lengthsuch that primary primers preferentially hybridize to them. However,when an undesired primer dimer product forms, it will hairpin on itselfvia its complementary 5′ ends and be unsuitable for continuedamplification.

FIG. 158 illustrates an exemplary PCR-PCR carryover prevention reactionto detect methylation in accordance with this aspect of the presentinvention. In this embodiment, genomic DNA or cfDNA is isolated andtreated with methyl-sensitive restriction endonucleases, e.g., Bsh1236I(CGACG) and/or HinP1I (GACGC), and UNG to completely digest unmethylatedDNA and prevent carryover (FIG. 158, step A). As shown in FIG. 158, stepB, methylated regions of interest are amplified using PCR in presence ofdUTP using locus-specific primers. In one embodiment, limited cycleamplification (12-20 cycles) is performed to maintain relative ratios ofdifferent amplicons being produced. In another embodiment, the regionsof interest are amplified using 20-40 cycles. Primers contain identical8-11 base tails to prevent primer dimers. The PCR products contain dU,allowing for carryover prevention (FIG. 158, step C). Optionally, thesample is aliquoted into 12, 24, 48, or 96 wells prior to PCR. Themethyl containing regions are amplified using nested or semi-nestedlocus-specific primers and an internal traditional TaqMan™ detectionassay. PCR products incorporate dU, allowing for carryover prevention.

FIG. 53 illustrates another PCR-qPCR carryover prevention reaction todetect methylation. Similar to the other embodiments of the presentinvention, genomic DNA or cfDNA is isolated and treated withmethyl-sensitive restriction endonucleases, e.g., Bsh1236I(CG{circumflex over ( )}CG) and UNG (37° C., 30-60 minutes) tocompletely digest unmethylated DNA and prevent carryover (FIG. 53, stepA). The digested DNA is bisulfite-treated to convert unmethylatedresidues to uracil thereby rendering the double stranded DNAnon-complementary. As shown in FIG. 53, step B, locus-specific primerscontaining a 3′ cleavable blocking group are hybridized in the presenceof BstU1 (CG{circumflex over ( )}CG) (filled triangles), which willcleave carryover DNA containing unmethylated residues. Once the primershybridize to their complementary target sequence, the blocking group isremoved. In this embodiment, the methyl-containing region of interest isamplified using PCR in the presence of dNTP. In this embodiment, ablocking oligonucleotide primer is used during amplification to limitformation of wild-type amplicon. As shown in FIG. 53, step C, the PCRproducts are unmethylated providing carryover protection.

As shown in FIG. 53, steps D and E, the PCR products are aliquot intoseparate wells for TaqMan™ detection using locus-specific primers thatare optionally nested or semi-nested to the primary set of locusspecific primers, and TaqMan™ probe (black bar). Optionally, a blockingoligonucleotide (thick black bar) can also be incorporated in thisreaction to limit formation of wild-type amplicons. In this embodiment,the TaqMan™ reaction is carried out in the presence of dUTPs, allowingfor carryover prevention.

Another aspect of the present invention is directed to a method foridentifying in a sample, one or more ribonucleic acid moleculescontaining a target ribonucleotide sequence differing fromribonucleotide sequences in other ribonucleic acid molecules in thesample due to alternative splicing, alternative transcript, alternativestart site, alternative coding sequence, alternative non-codingsequence, exon insertion, exon deletion, intron insertion,translocation, mutation, or other rearrangement at the genome level.This method involves providing a sample containing one or moreribonucleic acid molecules potentially containing a targetribonucleotide sequence differing from ribonucleotide sequences in otherribonucleic acid molecules, and contacting the sample with one or moreenzymes capable of digesting dU containing nucleic acid moleculespotentially present in the sample. One or more oligonucleotide primersare provided, each primer being complementary to the one or moreribonucleic acid molecules containing a target ribonucleotide sequence.The contacted sample is blended with the one or more oligonucleotideprimers, and a reverse-transcriptase to form a reverse-transcriptionmixture, and complementary deoxyribonucleic acid (cDNA) molecules aregenerated in the reverse transcription mixture. Each cDNA moleculecomprises a nucleotide sequence that is complementary to the targetribonucleotide sequence and contains dU. The method further involvesproviding one or more oligonucleotide primer sets, each primer setcomprising (a) a first oligonucleotide primer comprising a nucleotidesequence that is complementary to a portion of a cDNA nucleotidesequence adjacent to the target ribonucleotide sequence complement ofthe cDNA, and (b) a second oligonucleotide primer comprising anucleotide sequence that is complementary to a portion of an extensionproduct formed from the first oligonucleotide primer. The reversetranscription mixture containing the cDNA molecules is blended with theone or more oligonucleotide primer sets, and a polymerase to form apolymerase reaction mixture, and the polymerase chain reaction mixtureis subjected to one or more polymerase chain reaction cycles comprisinga denaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming one or more different primary extensionproducts. The method further involves providing one or moreoligonucleotide probe sets. Each probe set comprises (a) a firstoligonucleotide probe having a target sequence-specific portion, and (b)a second oligonucleotide probe having a target sequence-specificportion, wherein the first and second oligonucleotide probes of a probeset are configured to hybridize, in a base specific manner, adjacent toone another on a complementary primary extension product with a junctionbetween them. The primary extension products are contacted with a ligaseand the one or more oligonucleotide probe sets to form a ligationreaction mixture and the first and second probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligase reaction mixture. The ligated product sequencesin the sample are detected and distinguished thereby identifying thepresence of one or more ribonucleic acid molecules containing the targetribonucleotide sequence differing from ribonucleotide sequences in otherribonucleic acid molecules in the sample due to alternative splicing,alternative transcript, alternative start site, alternative codingsequence, alternative non-coding sequence, exon insertion, exondeletion, intron insertion, translocation, mutation, or otherrearrangement at the genome level

FIGS. 54-85 illustrate various embodiments of this aspect of the presentinvention.

FIG. 54 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to detect translocations at the mRNA level. An illustration oftranslocations between two genes is shown at the DNA level in FIG. 54,step A. Examples of the different fusion junctions between exons 1-b(mRNA fusion 1), 2-b (mRNA fusion 2), and 3-b (mRNA fusion 3) in mRNAsare illustrated (FIG. 54, step B). This method involves isolating mRNAfrom whole blood cells, exosomes, or circulating tumor cells (CTCs) andgenerating cDNA using reverse transcriptase and a primer complementaryto exon b. The generated cDNA is PCR amplified using forward primers toexons 1, 2, and 3 and the primer to exon b (FIG. 54, step B).Independent of translocation breakpoint, the primers will amplify thesmallest fragment containing the exon junction region. The variousproducts formed during PCR amplification are shown in FIG. 54, step C.

LDR is carried out using exon junction-specific ligation oligonucleotideprobes. The ligation probes can be designed to contain tag primerspecific portions (e.g., Ai, Ci′) suitable for subsequent detectionusing primers (Ai, Ci) and TaqMan™ probes (FIG. 54, steps C-D, leftpanel). Alternatively, the ligation probes can be designed to containUniTaq primer-specific (Ai, Ci′) and tag-specific portions (Bi′) (FIG.54, steps C-D, right panel). Following the formation of exon junctionspecific ligation product formation, the ligation products are PCRamplified and detected (FIG. 54, step D). When using tag-specificprimers (Ai, Ci) for amplifying LDR products, each TaqMan™ probe spansthe ligation junction, and can be scored individually. When usingUniTaq-specific primers (F1-Bi-Q-Ai, Ci), for amplifying LDR products,the same primer set scores for the given translocation, independent ofthe specific exon junction.

FIG. 55 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction todetect translocations at the mRNA level. In this embodiment, mRNA isisolated (FIG. 55, step A), and treated with UDG for carryoverprevention (FIG. 55, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. Taq polymerase is activated to perform limited cycle PCRamplification (12-20) to maintain relative ratios of different amplicons(FIG. 55, step B). The primers contain identical 8-11 base tails toprevent primer dimers. PCR products incorporate dUTP, allowing forcarryover prevention (FIG. 55, step C).

As shown in FIG. 55, step D, exon junction-specific ligationoligonucleotide probes containing primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 55, step D),and ligation products are aliquoted into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probe (F1-Q) which spans theligation junction (FIG. 55, step E). Treat samples with UDG forcarryover prevention, which also destroys original target amplicons(FIG. 55, step E). Only authentic LDR products will amplify, when usingPCR in presence of dUTP. Neither original PCR primers nor LDR probesamplify LDR products, providing additional carryover protection.

FIG. 56 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to detect translocations at the mRNA level. In this embodiment,mRNA is isolated (FIG. 56, step A), and treated with UDG for carryoverprevention (FIG. 56, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. Taq polymerase is activated to perform limited cycle PCRamplification (12-20) to maintain relative ratios of different amplicons(FIG. 56, step B). The primers contain identical 8-11 base tails toprevent primer dimers. PCR products incorporate dU, allowing forcarryover prevention (FIG. 56, step C).

As shown in FIG. 56, step D, exon junction-specific ligationoligonucleotide probes containing UniTaq primer-specific portions (Ai,Ci′) and tag portion (Bi′) suitable for subsequent PCR amplification anddetection, hybridize to nucleic acid sequences corresponding to thetarget mRNA molecule to be detected (FIG. 56, step D). Followingligation of the oligonucleotide probes, the sample is UDG treated toremove original target amplicons, allowing selective amplification anddetection of the ligation products using UniTaq-specific primers(F1-Bi-Q-Ai, Ci) as described supra (FIG. 56, steps E-F), therebyfacilitating the detection of the mRNA translocation mRNA fusion. Asshown in FIG. 56, steps E and F, the amplified ligation productsincorporate dUTP to allow for future carry over prevention.

FIG. 57 illustrates an example of a RT-PCR-qLDR carryover preventionreaction to detect translocations at the mRNA level. In this embodiment,mRNA is isolated (FIG. 57, step A), and treated with UDG for carryoverprevention (FIG. 57, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. Taq polymerase is activated to perform limited cycle PCRamplification (12-20) to maintain relative ratios of different amplicons(FIG. 57, step B). The primers contain identical 8-11 base tails toprevent primer dimers and universal primer-specific portions to enable asubsequent universal PCR amplification using biotin labeled primers toappend a 5′ biotin to the amplification products containing the regionof interest. PCR products incorporate dU, allowing for carryoverprevention (FIG. 57, step C). The biotinylated PCR products areimmobilized to a solid support and the region of interest is detectedusing exon junction-specific ligation probes as illustrated in FIG. 57,step D. In this embodiment, the exon junction-specific ligation probesof a ligation pair contain complementary tail sequences and an acceptoror donor group, respectively, capable of generating a detectable signalvia FRET when brought in close proximity to each other as describedsupra. Accordingly, following ligation (FIG. 57, step D), thecomplementary 5′ and 3′ tail ends of the ligation products hybridize toeach other bringing their respective donor and acceptor moieties inclose proximity to each other to generate a detectable FRET signal (FIG.57, step E).

FIG. 58 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to detect alternative splicing. FIG. 58, step A is anillustration of a gene with 5 exons shown at the DNA level, and FIG. 58,step B shows examples of normal (1-2-3a-4), and alternatively spliced(1-2-3b-4) variant mRNAs. This method involves isolating mRNA from wholeblood cells, exosomes, or CTCs, and generating cDNA using reversetranscriptase with a primer complementary to exon 4 as shown in FIG. 58,step B. The cDNA is PCR amplified using the exon 4 primer and a forwardprimer to exon 2, to generate amplicons of both normal and alternativesplice variants, if present. As shown in FIG. 58, step C, exonjunction-specific ligation oligonucleotide probes containing tag-primersequences (Ai, Ci′; left panel) or UniTaq primer and tag sequences (Ai,Bi′-Ci′; right panel) hybridize to their corresponding target sequencein the PCR products, and ligase covalently seals the twooligonucleotides together if there is perfect complementarity at thejunction. The ligation products are amplified and detected usingtag-specific primers (Ai, Ci) and TaqMan™ probes (F1-Q or F2-Q, FIG. 58,step D, left panel) or UniTaq primers (F1-Bi-Q-Ai, F2-Bi-Q-Ai, Ci, FIG.58, step D, right panel), as described supra.

FIG. 59 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction toquantify wild-type and alternatively spliced mRNA transcripts. FIG. 59,step A illustrates the wildtype transcript containing exon 3a (top) andalternatively spliced transcript containing exon 3b (bottom) to bedetected. This method involves isolating mRNA and treating with UDG forcarryover prevention (FIG. 59, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. Taq polymerase is activated to perform limited cycle PCRamplification (12-20) to maintain relative ratios of different amplicons(FIG. 59, step B). The primers contain identical 8-11 base tails toprevent primer dimers. PCR products incorporate dU, allowing forcarryover prevention (FIG. 59, step C). As shown in FIG. 59, step D,exon junction-specific ligation oligonucleotide probes containingtag-primer-specific portions (Ai, Ci═) suitable for subsequent PCRamplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 59, step D), and ligation products are aliquoted intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probes (F1-Q and F2-Q) which span the ligation junction (FIG. 59, stepE-F). The wild-type and alternative splice variant are quantified anddistinguished using real-time PCR and detecting the differently labeledTaqMan™ probes. Treat samples with UDG for carryover prevention, whichalso destroys original target amplicons (FIG. 59, step E). Onlyauthentic LDR products will amplify, when using PCR in presence of dUTP.Neither original PCR primers nor LDR probes amplify LDR products,providing additional carryover protection.

FIG. 60 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to quantify wild-type and alternatively spliced mRNAtranscripts. FIG. 60, step A illustrates the wildtype transcriptcontaining exon 3a (top) and alternatively spliced transcript containingexon 3b (bottom) to be detected. Steps A-D of this method areessentially the same as that described for FIG. 59, except that the exonjunction-specific ligation probes are designed to contain UniTaq primersequences (Ai, Ci′) and a UniTaq tag sequence (Bi′). Accordingly, inthis embodiment, the ligation products corresponding to the wild-typeand alternative splice variant are subsequently amplified, detected, andquantified using real time PCR with UniTaq-specific primers (F1-Bi-Q-Ai,Ci) as described above and illustrated in FIG. 60, steps E-G.

FIG. 61 illustrates a RT-PCR-qLDR carryover prevention reaction toquantify wild-type and alternatively spliced mRNA transcripts. FIG. 61,step A illustrates the wild-type transcript containing exon 3a (top) andalternatively spliced transcript containing exon 3b (bottom) to bedetected. This method involves isolating mRNA and treating with UDG forcarryover prevention (FIG. 61, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. The primers contain identical 8-11 base tails to prevent primerdimers and universal primer-specific portions to enable a subsequentuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest.PCR products incorporate dU, allowing for carryover prevention (FIG. 61,step C). The biotinylated PCR products are immobilized to a solidsupport and the region of interest is detected using exonjunction-specific ligation probes as illustrated in FIG. 61, step D. Inthis embodiment, the exon junction-specific ligation probes of aligation pair contain complementary tail sequences and an acceptor ordonor group, respectively, capable of generating a detectable signal viaFRET when brought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 61, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 61, step E).

FIG. 62 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction todetect low-level alternatively spliced transcripts. FIG. 62, step Aillustrates the wild-type transcript containing exon 3a (top) and thelow level alternatively spliced transcript containing exon 3b (bottom)to be detected. This method involves isolating mRNA and treating withUDG for carryover prevention (FIG. 62, step B). cDNA is generated using3′ transcript-specific primers (i.e. to exon 4) andreverse-transcriptase in the presence of dUTP. Taq polymerase isactivated to perform limited cycle PCR amplification (12-20) to maintainrelative ratios of different amplicons (FIG. 62, step B). In thisembodiment, a primer specific for the alternative splice variant (i.e.,exon 3b), and which does not hybridize to the wild-type variant (i.e.,exon 3a), is utilized to only generate amplification productscorresponding to the alternative splice variant. PCR productsincorporate dUTP, allowing for carryover prevention (FIG. 62, step C).As shown in FIG. 62, step D, exon junction-specific ligationoligonucleotide probes containing primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 62, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probe (F1-Q) which span theligation junction (FIG. 62, step E-F). The alternative splice variant isdetected during real-time PCR by the liberation of fluorescent group ofthe TaqMan™ probe. Samples are treated with UDG for carryoverprevention, which also destroys original target amplicons (FIG. 62, stepE). Only authentic LDR products are amplified, when using PCR inpresence of dUTP. Neither original PCR primers nor LDR probes amplifyLDR products, providing additional carryover protection.

FIGS. 63 and 64 illustrate similar RT-PCR-LDR-qPCR and RT-PCR-qLDRcarryover prevention reactions to detect low-level alternatively splicedtranscript as described and shown with respect to FIG. 62. In theembodiment of FIG. 63, the exon junction-specific ligation probes aredesigned to contain UniTaq primer sequences (Ai, Ci′) and a UniTaq tagsequence (Bi′). Accordingly, in this embodiment, the ligation productscorresponding to the alternative splice variant are subsequentlyamplified, detected, and quantified using real time PCR withUniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above and asillustrated in FIG. 63, steps E-G. In the embodiment of FIG. 64, theexon junction-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 64, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties (D, F2) in closeproximity to each other to generate a detectable FRET signal (FIG. 64,step E).

FIG. 65 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to detect alternative splicing. FIG. 65, step A shows anillustration of a gene with 3 exons, and an alternative start site andfirst exon at the DNA level. FIG. 65, step B shows examples of thenormal (1-2-3) and the alternative splice variant (1a-2-3) mRNAs. Thismethod involves isolating mRNA from whole blood cells, exosomes, orCTCs, and generating cDNA using reverse transcriptase with a primercomplementary to exon 2 as shown in FIG. 65, step B. The cDNA is PCRamplified using the exon 2 primer and a forward primer that iscomplementary to either exon 1 or exon 1a to generate amplicons of bothsplice variants. As shown in FIG. 65, step C, exon junction-specificligation oligonucleotide probes containing tag-primer sequences (Ai,Ci′; left panel) or UniTaq primer and tag sequences (Ai, Bi′-Ci′; rightpanel) hybridize to their corresponding target sequence in the PCRproducts, and ligase covalently seals the two oligonucleotides togetherif there is perfect complementarity at the junction. The ligationproducts are amplified and detected using tag-specific primers (Ai, Ci)and TaqMan™ probes (F1-Q or F2-Q; FIG. 58, step D, left panel) or UniTaqprimers (F1-Bi-Q-Ai, F2-Bi-Q-Ai, and Ci, FIG. 58, step D, right panel),as described supra.

FIG. 66 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction toquantify transcripts containing the wild-type and an alternativetranscription start site. FIG. 66, step A illustrates the wildtypetranscript containing exon 1 (top) and the alternative transcript havingexon 1a as the start site (bottom). This method involves isolating mRNAand treating with UDG for carryover prevention (FIG. 66, step B). cDNAis generated using 3′ transcript-specific primers andreverse-transcriptase in the presence of dUTP. The cDNA is PCR amplifiedusing the exon 2-specific primer and a forward primer that iscomplementary to either exon 1 or exon 1a to generate amplicons of bothsplice variants. Limited PCR amplification (12-20 cycles) is performedto maintain relative ratios of different amplicons (FIG. 66, step B). Inanother embodiment, regions of interest are amplified using 20-40 PCRcycles. The primers contain identical 8-11 base tails to prevent primerdimers. PCR products incorporate dU, allowing for carryover prevention(FIG. 66, step C). As shown in FIG. 66, step D, exon junction-specificligation oligonucleotide probes containing tag-primer-specific portions(Ai, Ci′) suitable for subsequent TaqMan™ PCR amplification, hybridizeto their corresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 66, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probes (F1-Q and F2-Q) which spanthe ligation junction (FIG. 66, step E-F). The wild-type and alternativetranscript start site variant are quantified and distinguished usingreal-time PCR and detecting the differently labeled TaqMan™ probes.Samples are treated with UDG for carryover prevention, which alsodestroys original target amplicons (FIG. 66, step E). Only authentic LDRproducts amplify, when using PCR in presence of dUTP. Neither originalPCR primers nor LDR probes amplify LDR products, providing additionalcarryover protection.

FIG. 67 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to quantify wild-type and alternatively spliced mRNAtranscripts. FIG. 67, step A illustrates the wildtype transcriptcontaining exon 1 (top) and the alternative transcript having exon 1a asthe start site (bottom). Steps A-D of this method are essentially thesame as that described for FIG. 66, except that the exonjunction-specific ligation probes are designed to contain UniTaq primersequences (Ai, Ci′) and a UniTaq tag sequence (Bi′). Accordingly, inthis embodiment, the ligation products corresponding to the wildtype andvariant transcripts are subsequently amplified, detected, and quantifiedusing real time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) asdescribed above and illustrated in FIG. 67, steps E-F.

FIG. 68 illustrates RT-PCR-qLDR carryover prevention reaction toquantify wild-type and alternatively spliced mRNA transcripts. FIG. 68,step A illustrates the wildtype transcript containing exon 1 (top) andthe alternative transcript having exon 1a as the start site (bottom).This method involves isolating mRNA and treating with UDG for carryoverprevention (FIG. 68, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. The cDNA is PCR amplified using the exon 2 primer and a forwardprimer that is complementary to either exon 1 or exon 1a to generateamplicons of both splice variants. The primers contain identical 8-11base tails to prevent primer dimers and universal primer-specificportions to enable a subsequent universal PCR amplification using biotinlabeled primers to append a 5′ biotin to the amplification productscontaining the region of interest. PCR products incorporate dU, allowingfor carryover prevention (FIG. 68, step C). The biotinylated PCRproducts are immobilized to a solid support and the region of interestis detected using exon junction-specific ligation probes as illustratedin FIG. 68, step D. In this embodiment, the exon junction-specificligation probes of a ligation pair contain complementary tail sequencesand an acceptor or donor group, respectively, capable of generating adetectable signal via FRET when brought in close proximity to each otheras described supra. Accordingly, following ligation (FIG. 68, step D),the complementary 5′ and 3′ tail ends of the ligation products hybridizeto each other bringing their respective donor and acceptor moieties inclose proximity to each other to generate a detectable FRET signal (FIG.68, step E).

FIG. 69 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction todetect low-level of the alternative start site transcript. FIG. 69, stepA illustrates the wildtype transcript containing exon 1 (top) and thealternative transcript having exon 1a as the start site (bottom). Thismethod involves isolating mRNA and treating with UDG for carryoverprevention (FIG. 69, step B). cDNA is generated using 3′transcript-specific primers and reverse-transcriptase in the presence ofdUTP. Taq polymerase is activated to perform limited cycle PCRamplification (12-20) to maintain relative ratios of different amplicons(FIG. 69, step B). In this embodiment, a primer specific for thealternative transcript (i.e., exon 1a ), which does not hybridize to thewildtype variant (i.e., exon 1), is used to only generate amplificationproducts corresponding to the alternative transcript. PCR productsincorporate dUTP, allowing for carryover prevention (FIG. 69, step C).As shown in FIG. 69, step D, exon junction-specific ligationoligonucleotide probes containing primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 69, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probe (F1-Q) which span theligation junction (FIG. 69, step E-F). The alternative transcript isdetected during real-time PCR by the liberation of fluorescent group ofthe TaqMan™ probe. Samples are treated with UDG for carryoverprevention, which also destroys original target amplicons (FIG. 69, stepE). Only authentic LDR products are amplified, when using PCR inpresence of dUTP. Neither original PCR primers nor LDR probes amplifyLDR products, providing additional carryover protection.

FIGS. 70 and 71 illustrate similar RT-PCR-LDR-qPCR and RT-PCR-qLDRcarryover prevention reactions to detect low-level alternative startsite transcript as described and shown with respect to FIG. 69 (stepsA-C), where only primers specific for the amplification of thealternative transcript are utilized. In the embodiment of FIG. 70, theexon junction-specific ligation probes are designed to contain UniTaqprimer sequences (Ai, Ci′) and a UniTaq tag sequence (Bi′). Accordingly,in this embodiment, ligation products corresponding to the alternativestart site transcript are subsequently amplified, detected, andquantified using real time PCR with UniTaq-specific primers (F1-Bi-Q-Ai,Ci) as described above and as illustrated in FIG. 70, steps E-G. In theembodiment of FIG. 71, the exon junction-specific ligation probes of aligation pair contain complementary tail sequences and an acceptor ordonor group, respectively, capable of generating a detectable signal viaFRET when brought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 71, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties (D, F2) in closeproximity to each other to generate a detectable FRET signal (FIG. 71,step E).

FIG. 72 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to detect exon deletion. FIG. 72, step A shows an illustrationof a gene with 5 exons and 4 introns at the DNA level. FIG. 72, step Bshows examples of the wild-type transcript containing exons 1-5 (top)and the alternative transcript where exon 4 is deleted (bottom, i.e.,exons 1-3, and 5). This method involves isolating mRNA from whole bloodcells, exosomes, or CTCs, and generating cDNA using reversetranscriptase with a primer complementary to exon 5 as shown in FIG. 72,step B. The cDNA is PCR amplified using the exon 5 primer in conjunctionwith forward primers complementary to exon 3 and exon 4 to generateamplicons of both wild-type and the deletion variant. As shown in FIG.72, step C, exon junction-specific ligation oligonucleotide probescontaining tag-primer sequences (Ai, Ci′; left panel) or UniTaq primerand tag sequences (Ai, Bi′-Ci′; right panel), hybridize to theircorresponding target sequence in the PCR products, and ligase covalentlyseals the two oligonucleotides together if there is perfectcomplementarity at the junction. The ligation products are amplified anddetected using tag-specific primers (Ai, Ci), and TaqMan™ probes (F1-Qor F2-Q; FIG. 72, step D, left panel) or UniTaq primers (F1-Bi-Q-Ai,F2-Bi-Q-Ai, and Ci, FIG. 72, step D, right panel), as described supra.

FIG. 73 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction toquantify wild-type transcripts and transcripts having an exon deletion.FIG. 73, step A illustrates the wild-type transcript containing exons1-5 (top) and the alternative transcript having only exons 1-3 and 5,where exon 4 is missing (bottom). This method involves isolating mRNAand treating with UDG for carryover prevention (FIG. 73, step B). cDNAis generated using a 3′ transcript-specific primer (e.g., exon 5specific primer) and reverse-transcriptase in the presence of dUTP. ThecDNA is PCR amplified using the exon 5 primer in conjunction withforward primers complementary to exon 3 and exon 4 to generate ampliconsof both wild-type and the deletion variant. Limited PCR amplification(12-20 cycles) is performed to maintain relative ratios of differentamplicons (FIG. 73, step B). In another embodiment, the regions ofinterest are amplified using 20-40 PCR cycles. The primers containidentical 8-11 base tails to prevent primer dimers. PCR productsincorporate dU, allowing for carryover prevention (FIG. 73, step C). Asshown in FIG. 73, step D, exon junction-specific ligationoligonucleotide probes containing primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 73, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probes (F1-Q and F2-Q) which spanthe ligation junction (FIG. 73, step E-F). The wild-type and deletionvariant are quantified and distinguished using real-time PCR anddetecting the differently labeled TaqMan™ probes. Samples are treatedwith UDG for carryover prevention, which also destroys original targetamplicons (FIG. 73, step E). Only authentic LDR products amplify, whenusing PCR in presence of dUTP. Neither original PCR primers nor LDRprobes amplify LDR products, providing additional carryover protection.

FIG. 74 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to quantify wildtype transcripts and transcripts having an exondeletion. FIG. 74, step A illustrates the wildtype transcript containingexons 1-5 (top) and the alternative transcript having only exons 1-3 and5, where exon 4 is missing (bottom). Steps A-D of this method areessentially the same as that described for FIG. 73, except that the exonjunction-specific ligation probes are designed to contain UniTaq primersequences (Ai, Ci′) and a UniTaq tag sequence (Bi′). Accordingly, inthis embodiment, the ligation products corresponding to the wildtype andvariant transcripts are subsequently amplified, detected, and quantifiedusing real time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) asdescribed above and illustrated in FIG. 74, steps E-G.

FIG. 75 illustrates RT-PCR-qLDR carryover prevention reaction toquantify wildtype transcripts and transcripts having an exon deletion.FIG. 75, step A illustrates the wildtype transcript containing exons 1-5(top) and the alternative transcript having only exons 1-3 and 5, whereexon 4 is missing (bottom). This method involves isolating mRNA andtreating with UDG for carryover prevention (FIG. 75, step B). cDNA isgenerated using a 3′ transcript-specific primer (e.g., exon 5 specificprimer) and reverse-transcriptase in the presence of dUTP. The cDNA isPCR amplified using the exon 5 primer in conjunction with forwardprimers complementary to exon 3 and exon 4 to generate amplicons of bothwildtype and the deletion variant. The primers contain identical 8-11base tails to prevent primer dimers and universal primer-specificportions to enable a subsequent universal PCR amplification using biotinlabeled primers to append a 5′ biotin to the amplification productscontaining the region of interest. PCR products incorporate dU, allowingfor carryover prevention (FIG. 75, step C). The biotinylated PCRproducts are immobilized to a solid support and the region of interestis detected using exon junction-specific ligation probes as illustratedin FIG. 75, step D. In this embodiment, the exon junction-specificligation probes of a ligation pair contain complementary tail sequencesand an acceptor or donor group, respectively, capable of generating adetectable signal via FRET when brought in close proximity to each otheras described supra. Accordingly, following ligation (FIG. 75, step D),the complementary 5′ and 3′ tail ends of the ligation products hybridizeto each other bringing their respective donor and acceptor moieties inclose proximity to each other to generate a detectable FRET signal (FIG.75, step E).

FIG. 76 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction todetect low-level transcripts having an exon deletion. FIG. 76, step Aillustrates the wildtype transcript containing exons 1-5 (top) and thealternative transcript having only exons 1-3 and 5, where exon 4 ismissing (bottom). This method involves isolating mRNA and treating withUDG for carryover prevention (FIG. 76, step B). cDNA is generated usinga 3′ transcript-specific primer (e.g., an exon 5 specific primer) andreverse-transcriptase in the presence of dUTP. The cDNA is PCR amplifiedusing the exon 5 primer in conjunction with a forward primercomplementary to exon 3 and a blocking oligonucleotide. The blockingoligonucleotide hybridizes to exon 4 in the wild-type transcript,thereby preventing amplification of wild-type transcripts. Accordingly,only amplification products corresponding to the deletion transcript aregenerated. PCR products incorporate dUTP, allowing for carryoverprevention (FIG. 76, step C). As shown in FIG. 76, step D, exonjunction-specific ligation oligonucleotide probes containing tagprimer-specific portions (Ai, Ci′) suitable for subsequent PCRamplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 76 step D), and ligation products are aliquot intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probe (F1-Q) which span the ligation junction (FIG. 76, step E-F). Thedeletion transcript is detected during real-time PCR by the liberationof fluorescent group of the TaqMan™ probe. Samples are treated with UDGfor carryover prevention, which also destroys original target amplicons(FIG. 76, step E). Only authentic LDR products are amplified, when usingPCR in presence of dUTP. Neither original PCR primers nor LDR probesamplify LDR products, providing additional carryover protection.

FIGS. 77 and 78 illustrate similar RT-PCR-LDR-qPCR and RT-PCR-qLDRcarryover prevention reactions to detect low-level deletion transcriptsas described and shown with respect to FIG. 76. In the embodiment ofFIG. 77, the exon junction-specific ligation probes are designed tocontain UniTaq primer sequences (Ai, Ci′) and a UniTaq tag sequence(Bi′). Accordingly, in this embodiment, ligation products correspondingto the deletion transcript are subsequently amplified, detected, andquantified using real time PCR with UniTaq-specific primers (F1-Bi-Q-Ai,Ci) as described above and as illustrated in FIG. 77, steps E-G. In theembodiment of FIG. 78, the exon junction-specific ligation probes of aligation pair contain complementary tail sequences and an acceptor ordonor group, respectively, capable of generating a detectable signal viaFRET when brought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 78, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties (D, F2) in closeproximity to each other to generate a detectable FRET signal (FIG. 78,step E).

FIG. 79 illustrates an overview of RT-PCR-LDR-qPCR carryover preventionreaction to detect alternative splicing with intron insertion. FIG. 79,step A shows an illustration of a gene with 5 exons and 4 introns at theDNA level. FIG. 79, step B shows examples of the wildtype transcriptcontaining exons 1-5 (top) and the alternatively spliced transcriptcontaining exons 1-5 with an intron i1 insertion (bottom). This methodinvolves isolating mRNA from whole blood cells, exosomes, or CTCs, andgenerating cDNA using reverse transcriptase with a primer complementaryto exon 2 as shown in FIG. 79, step B. The cDNA is PCR amplified usingthe exon 2-specific primer in conjunction with a forward primer to exon1, to generate amplicons of both wild-type and the intron insertionvariant. As shown in FIG. 79, step C, exon junction-specific ligationoligonucleotide probes containing tag-primer sequences (Ai, Ci′; leftpanel) or UniTaq primer and tag sequences (Ai, Bi′-Ci′; right panel)hybridize to their corresponding target sequence in the PCR products,and ligase covalently seals the two oligonucleotides together if thereis perfect complementarity at the junction. The ligation products areamplified and detected using tag-specific primers (Ai, Ci), and TaqMan™probes (F1-Q or F2-Q; FIG. 79, step D, left panel) or UniTaq primers(F1-Bi-Q-Ai, F2-Bi-Q-Ai, and Ci, FIG. 79, step D, right panel), asdescribed supra.

FIG. 80 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction toquantify wildtype transcripts and alternatively spliced transcriptscontaining an intron insertion. FIG. 80, step B shows examples of thewildtype transcript containing exons 1-5 (top) and the alternativelyspliced transcript containing exons 1-5 with an intron i1 insertion(bottom). This method involves isolating mRNA and treating with UDG forcarryover prevention (FIG. 80, step B). cDNA is generated using a 3′transcript-specific primer (e.g., exon 2 specific primer) andreverse-transcriptase in the presence of dUTP. The cDNA is PCR amplifiedusing the exon 2-specific primer in conjunction with a forward primer toexon 1 to generate amplicons of both wildtype and the intron insertionvariant. Limited PCR amplification (12-20 cycles) is performed tomaintain relative ratios of different amplicons (FIG. 80, step B). Inanother embodiment, the regions of interest are amplified using 20-40cycles. The primers contain identical 8-11 base tails to prevent primerdimers. PCR products incorporate dU, allowing for carryover prevention(FIG. 80, step C). As shown in FIG. 80, step D, exon junction-specificligation oligonucleotide probes containing tag primer-specific portions(Ai, Ci′) suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 80, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probes (F1-Q and F2-Q) which spanthe ligation junction (FIG. 80, step E-F). The wild-type and insertionvariant are quantified and distinguished using real-time PCR anddetecting the differently labeled TaqMan™ probes. Samples are treatedwith UDG for carryover prevention, which also destroys original targetamplicons (FIG. 80, step E). Only authentic LDR products amplify, whenusing PCR in presence of dUTP. Neither original PCR primers nor LDRprobes amplify LDR products, providing additional carryover protection.

FIG. 81 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to quantify wildtype transcripts and alternatively splicedtranscripts containing an intron insertion. FIG. 81, step B showsexamples of the wildtype transcript containing exons 1-5 (top) and thealternatively spliced transcript containing exons 1-5 with an intron i1insertion (bottom). Steps A-D of this method are essentially the same asthat described for FIG. 80, except that the exon junction-specificligation probes are designed to contain UniTaq primer sequences (Ai,Ci′) and a UniTaq tag sequence (Bi′). Accordingly, in this embodiment,the ligation products corresponding to the wildtype and varianttranscripts are subsequently amplified, detected, and quantified usingreal time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as describedabove and illustrated in FIG. 81, steps E-G.

FIG. 82 illustrates RT-PCR-qLDR carryover prevention reaction toquantify wildtype transcripts and alternatively spliced transcriptscontaining an intron insertion. FIG. 82, step B shows examples of thewildtype transcript containing exons 1-5 (top) and the alternativelyspliced transcript containing exons 1-5 with an intron i1 insertion(bottom). This method involves isolating mRNA and treating with UDG forcarryover prevention (FIG. 82, step B). cDNA is generated using a 3′transcript-specific primer (e.g., exon 2 specific primer) andreverse-transcriptase in the presence of dUTP. The cDNA is PCR amplifiedusing the exon 2-specific primer in conjunction with a forward primer toexon 1 to generate amplicons of both wildtype and the intron insertionvariant. The primers contain identical 8-11 base tails to prevent primerdimers and universal primer-specific portions to enable a subsequentuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest.PCR products incorporate dU, allowing for carryover prevention (FIG. 82,step C). The biotinylated PCR products are immobilized to a solidsupport and the region of interest is detected using exonjunction-specific ligation probes as illustrated in FIG. 82, step D. Inthis embodiment, the exon junction-specific ligation probes of aligation pair contain complementary tail sequences and an acceptor ordonor group, respectively, capable of generating a detectable signal viaFRET when brought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 82, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 82, step E).

FIG. 83 illustrates a RT-PCR-LDR-qPCR carryover prevention reaction todetect low-level transcripts containing an intron insertion. FIG. 83,step B shows examples of the wildtype transcript containing exons 1-5(top) and the alternatively spliced transcript containing exons 1-5 withan intron i1 insertion (bottom). This method involves isolating mRNA andtreating with UDG for carryover prevention (FIG. 83, step B). cDNA isgenerated using a 3′ transcript-specific primer (e.g., an exon2-specific primer) and reverse-transcriptase in the presence of dUTP.The cDNA is PCR amplified using the exon 2 primer in conjunction with anintron specific forward primer. The intron specific primer does notamplify the wildtype transcript, thus only transcript containing theintron i1 insertion are amplified. PCR products incorporate dU, allowingfor carryover prevention (FIG. 83, step C). As shown in FIG. 83, step D,exon junction-specific ligation oligonucleotide probes containingprimer-specific portions (Ai, Ci′) suitable for subsequent PCRamplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 83, step D), and ligation products are aliquot intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probe (F1-Q) which span the ligation junction (FIG. 83, step E-F). Thetranscript containing the intron insertion is detected during real-timePCR by the liberation of fluorescent group of the TaqMan™ probe. Samplesare treated with UDG for carryover prevention, which also destroysoriginal target amplicons (FIG. 83, step E). Only authentic LDR productsare amplified, when using PCR in presence of dUTP. Neither original PCRprimers nor LDR probes amplify LDR products, providing additionalcarryover protection.

FIGS. 84 and 85 illustrate similar RT-PCR-LDR-qPCR and RT-PCR-qLDRcarryover prevention reactions to detect low-level intron insertiontranscripts as described and shown with respect to FIG. 83. In theembodiment of FIG. 84, the exon junction-specific ligation probes aredesigned to contain UniTaq primer sequences (Ai, Ci′) and a UniTaq tagsequence (Bi′). Accordingly, in this embodiment, ligation productscorresponding to the intron insertion transcript are subsequentlyamplified, detected, and quantified using real time PCR withUniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above and asillustrated in FIG. 84, steps E-G. In the embodiment of FIG. 85, theexon junction-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 85, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties (D, F1) in closeproximity to each other to generate a detectable FRET signal (FIG. 85,step E).

The methods of the present invention are suitable for quantifying orenumerating the amount of the one or more target nucleotide sequences ina sample. For example, the methods of the present invention can beutilized to enumerate the relative copy number of one or more targetnucleic acid molecules in a sample as illustrated in FIGS. 86-91.

FIG. 86 illustrates PCR-LDR carryover prevention reaction to enumerateDNA copy number. This method involves isolating DNA from CTCs,tumor-specific exosomes, or another biological sample, and treating withUDG for carryover prevention (FIG. 86, step B). Chromosomal regions ofinterest are amplified using limited cycle PCR (12-20 cycles) tomaintain relative ratios of different amplicons (FIG. 86, step B). Inanother embodiment, the chromosomal regions of interest are amplifiedusing 20-40 cycles. For accurate enumeration, the sample is dispersedinto 12, 24, 48, or 96 wells prior to PCR amplification. The primerscontain identical 8-11 base tails to prevent primer dimers. PCR productsincorporate dU, allowing for carryover prevention (FIG. 86, step C). Asshown in FIG. 86, step D, locus-specific ligation oligonucleotide probescontaining primer-specific portions (Ai, Ci′) suitable for subsequentPCR amplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 86, step D), and ligation products are aliquot intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probe (F1-Q) which span the ligation junction (FIG. 86, step E-F). TheDNA copy number is determined based on the Poisson distribution ofsignal in the different wells or chambers. Samples are treated with UDGfor carryover prevention, which also destroys original target amplicons(FIG. 86, step E). Only authentic LDR products will amplify, when usingPCR in presence of dUTP. Neither original PCR primers nor LDR probesamplify LDR products, providing additional carryover protection.

FIG. 87 illustrates another PCR-LDR-qPCR carryover prevention reactionto enumerate DNA copy number. This method involves essentially the samesteps (i.e., steps A-D) as the method illustrated in FIG. 86; however,in this embodiment, the locus specific ligation probes are designed tocontain UniTaq primer sequences (Ai, Ci′) and a UniTaq tag sequence(Bi′). Accordingly, in this embodiment, the ligation products aresubsequently amplified, detected, and quantified using real time PCRwith UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above andillustrated in FIG. 87, steps E-G. Copy number is determined based onthe Poisson distribution of signal in the different wells or chambers.

FIG. 88 also illustrates PCR-qLDR carryover prevention reaction toenumerate DNA copy number. This method involves isolating DNA from CTCs,tumor-specific exosomes, or another biological sample, and treating withUDG for carryover prevention (FIG. 88, step B). Chromosomal regions ofinterest are amplified using limited cycle PCR (12-20 cycles) tomaintain relative ratios of different amplicons (FIG. 88, step B). Inanother embodiment, the chromosomal regions of interest are amplifiedusing 20-40 cycles. For accurate enumeration, the sample is dispersedinto 12, 24, 48, or 96 wells prior to PCR amplification. The primerscontain identical 8-11 base tails to prevent primer dimers and universalprimer-specific portions to enable a subsequent universal PCRamplification using biotin labeled primers to append a 5′ biotin to theamplification products containing the region of interest. PCR productsincorporate dU, allowing for carryover prevention (FIG. 88, step C). Thebiotinylated PCR products are immobilized to a solid support and theregion of interest is detected using locus-specific ligation probes asillustrated in FIG. 88, step D. In this embodiment, the exonjunction-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 88, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 88, step E).Copy number is determined based on the Poisson distribution of signal inthe different wells or chambers.

FIG. 89 illustrates RT-PCR-LDR-qPCR carryover prevention reaction toenumerate RNA copy number. This method involves isolating RNA from wholeblood cells, exosomes, CTCs, or another biological sample, and treatingwith UDG for carryover prevention (FIG. 89, step B). cDNA is generatedusing 3′ transcript-specific primers and reverse-transcriptase in thepresence of dUTP. Taq polymerase is activated to perform limited cyclePCR amplification (12-20) to maintain relative ratios of differentamplicons (FIG. 89, step B). For accurate enumeration, the sample isdispersed into 12, 24, 48, or 96 wells prior to PCR amplification. Theprimers contain identical 8-11 base tails to prevent primer dimers. PCRproducts incorporate dU, allowing for carryover prevention (FIG. 89,step C). As shown in FIG. 89, step D, locus-specific ligationoligonucleotide probes containing tag primer-specific portions (Ai, Ci′)suitable for subsequent PCR amplification, hybridize to theircorresponding target sequence in a base-specific manner. Ligasecovalently seals the two oligonucleotides together (FIG. 89, step D),and ligation products are aliquot into separate wells for detectionusing tag-primers (Ai, Ci) and TaqMan™ probe (F1-Q) which span theligation junction (FIG. 89, step E-F). The RNA copy number is quantifiedusing real-time PCR based on the Poisson distribution of signal in thedifferent wells or chambers. Samples are treated with UDG for carryoverprevention, which also destroys original target amplicons (FIG. 89, stepE). Only authentic LDR products will amplify when using PCR in presenceof dUTP. Neither original PCR primers nor LDR probes amplify LDRproducts, providing additional carryover protection.

FIG. 90 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to enumerate RNA copy number. For accurate enumeration, thesample is dispersed into 12, 24, 48, or 96 wells prior to PCRamplification. This method involves essentially the same steps (i.e.,steps A-D) as the method illustrated in FIG. 89; however, in thisembodiment, the locus specific ligation probes are designed to containUniTaq primer sequences (Ai, Ci′) and a UniTaq tag sequence (Bi′).Accordingly, in this embodiment, the ligation products are subsequentlyamplified, detected, and quantified using real time PCR withUniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above andillustrated in FIG. 90, steps E-G. Copy number is determined based onthe Poisson distribution of signal in the different wells or chambers.

FIG. 91 illustrates RT-PCR-qLDR carryover prevention reaction toenumerate RNA copy number. This method involves isolating RNA from wholeblood cells, exosomes, CTCs, or another biological sample, and treatingwith UDG for carryover prevention (FIG. 91, step B). For accurateenumeration, the sample is dispersed into 12, 24, 48, or 96 wells priorto PCR amplification. cDNA is generated using 3′ transcript-specificprimers and reverse-transcriptase in the presence of dUTP. Taqpolymerase is activated to perform limited cycle PCR amplification(12-20) to maintain relative ratios of different amplicons (FIG. 91,step B). The primers contain identical 8-11 base tails to prevent primerdimers and universal primer-specific portions to enable a subsequentuniversal PCR amplification using biotin labeled primers to append a 5′biotin to the amplification products containing the region of interest.PCR products incorporate dU, allowing for carryover prevention (FIG. 91,step C). The biotinylated PCR products are immobilized to a solidsupport and the region of interest is detected using locus-specificligation probes as illustrated in FIG. 91, step D. In this embodiment,the exon junction-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 91, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 91, step E).Copy number is determined based on the Poisson distribution of signal inthe different wells or chambers. Another aspect of the present inventionis directed to a method for identifying, in a sample, one or moremicro-ribonucleic acid (miRNA) molecules containing a targetmicro-ribonucleotide sequence differing from micro-ribonucleotidesequences in other miRNA molecules in the sample by one or more bases.This method involves providing a sample containing one or more miRNAmolecules potentially containing the target micro-ribonucleotidesequence differing from micro-ribonucleotide sequences in other miRNAmolecules in the sample by one or more bases, and contacting the samplewith one or more enzymes capable of digesting dU containing nucleic acidmolecules potentially present in the sample. One or more oligonucleotideprimer sets are provided, each primer set comprising (a) a firstoligonucleotide primer having a 5′ stem-loop portion, a blocking group,an internal primer-specific portion within the loop region, and a 3′nucleotide sequence portion that is complementary to a 3′ portion of themiRNA molecule containing the target micro-ribonucleotide sequence, (b)a second oligonucleotide primer having a 3′ nucleotide sequence portionthat is complementary to a complement of the 5′ end of the miRNAmolecule containing the target micro-ribonucleotide sequence, and a 5′primer-specific portion, (c) a third oligonucleotide primer comprising anucleotide sequence that is the same as the internal primer-specificportion of the first oligonucleotide primer, and (d) a fourtholigonucleotide primer comprising a nucleotide sequence that is the sameas the 5′ primer-specific portion of the second oligonucleotide primer.The contacted sample is blended with the one or more firstoligonucleotide primers of a primer set, a deoxynucleotide mix includingdUTP, and a reverse transcriptase to form a reverse transcriptionreaction mixture. The first oligonucleotide primer hybridizes to themiRNA molecule containing the target micro-ribonucleotide sequence, ifpresent in the sample, and the reverse transcriptase extends the 3′ endof the hybridized first oligonucleotide primer to generate an extendedfirst oligonucleotide primer comprising the complement of the miRNAmolecule containing the target micro-ribonucleotide sequence. The methodfurther involves blending the reverse transcription reaction mixturewith the second, third, and fourth oligonucleotide primers of the primerset to form a polymerase reaction mixture under conditions effective forthe one or more second oligonucleotide primers of a primer set tohybridize to the region of the extended first oligonucleotide primercomprising the complement of the miRNA molecule containing the targetmicro-ribonucleotide sequence and extend to generate a primary extensionproduct comprising the 5′ primer-specific portion, a nucleotide sequencecorresponding to the target micro-ribonucleotide sequence of the miRNAmolecule, and the complement of the internal primer-specific portion.The polymerase chain reaction mixture is subjected to one or morepolymerase chain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby forming aplurality of primary extension products. The method further involvesblending the plurality of primary extension products with a ligase andone or more oligonucleotide probe sets to form a ligation reactionmixture. Each oligonucleotide probe set comprises (a) a firstoligonucleotide probe having a target-specific portion, and (b) a secondoligonucleotide probe having a target-specific portion and a portioncomplementary to a primary extension product, wherein the first andsecond oligonucleotide probes of a probe set are configured tohybridize, in a base specific manner, adjacent to one another oncomplementary target-specific portions of a primary extension productwith a junction between them. The first and second oligonucleotideprobes of the one or more oligonucleotide probe sets are ligatedtogether to form ligated product sequences in the ligation reactionmixture, and the ligated product sequences in the sample are detectedand distinguished thereby identifying one or more miRNA moleculescontaining a target micro-ribonucleotide sequence differing frommicro-ribonucleotide sequences in other miRNA molecules in the sample byone or more bases.

FIG. 92 illustrates PCR-LDR carryover prevention reaction to quantifymiRNA. This method involves isolating RNA from exosomes or anotherbiological sample, and treating with UDG for carryover prevention (FIG.92, step B). An oligonucleotide primer having a portion that iscomplementary to the 3′ end of the target miRNA, and containing astem-loop, tag (Tj), and blocking group (filled circle) is hybridized tothe 3′ end of the target miRNA. The 3′ end of the oligonucleotide primeris extended using reverse transcriptase (filled diamond) in the presenceof dUTP (FIG. 92, step B). Taq polymerase is activated to performlimited cycle PCR amplification (12-20) using a bridge primer comprisinga sequence that is complementary to a portion of the reverse transcribedmiRNA and an upstream primer-specific sequence portion (Ti), and tag(Ti, Tj) primers as shown in FIG. 92, step B. Primers contain identical8-11 base tails to prevent primer dimers. Optionally, the sample can bealiquot into 12, 24, 48, or 96 wells prior to PCR. PCR productsincorporate dUTP, allowing for carryover prevention (FIG. 92, step C).As shown in FIG. 92, step D, miRNA sequence-specific ligation probescontaining primer-specific portions (Ai, Ci′) suitable for subsequentPCR amplification, hybridize to their corresponding target sequence inthe PCR products in a base-specific manner. Ligase covalently seals thetwo oligonucleotides together (FIG. 92, step D), and ligation productsare aliquot into separate wells for detection using tag-primers (Ai, Ci)and TaqMan™ probe (F1-Q) which spans the ligation junction (FIG. 92,step E-F). The presence of the miRNA in the sample is quantified usingreal-time PCR based on the detection of the liberated label of theTaqMan™ probe. Samples are treated with UDG for carryover prevention,which also destroys original target amplicons (FIG. 92, step E). Onlyauthentic LDR products will amplify, when using PCR in presence of dUTP.Neither original PCR primers nor LDR probes amplify LDR products,providing additional carryover protection.

FIG. 93 illustrates another RT-PCR-LDR-qPCR carryover preventionreaction to quantify miRNA. This method involves essentially the samesteps (i.e., steps A-D) as the method illustrated in FIG. 92; however,in this embodiment, the miRNA specific ligation probes are designed tocontain UniTaq primer sequences (Ai, Ci′) and a UniTaq tag sequence(Bi′). Accordingly, in this embodiment, the ligation products aresubsequently amplified, detected, and quantified using real time PCRwith UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above andillustrated in FIG. 93, steps E-G.

FIG. 94 also illustrates RT-PCR-qLDR carryover prevention reaction toquantify miRNA. This method involves isolating RNA from exosomes oranother biological sample, and treating with UDG for carryoverprevention (FIG. 94, step B). An oligonucleotide primer having a portionthat is complementary to the 3′ end of the target miRNA, and containinga stem-loop, tag (Tj), and blocking group (filled circle) is hybridizedto the 3′ end of the target miRNA. The 3′ end of the oligonucleotideprimer is extended using reverse transcriptase (filled diamond) in thepresence of dUTP (FIG. 94, step B). Taq polymerase is activated toperform limited cycle PCR amplification (12-20) using a bridge primercomprising a sequence that is complementary to a portion of the reversetranscribed miRNA and an upstream primer-specific sequence portion (Ti),and tag (Ti, Tj) primers as shown in FIG. 94, step B. The primerscontain identical 8-11 base tails to prevent primer dimers and universalprimer-specific portions to enable a subsequent universal PCRamplification using biotin labeled primers to append a 5′ biotin to theamplification products containing the region of interest. PCR productsincorporate dU, allowing for carryover prevention (FIG. 94, step C). Thebiotinylated PCR products are immobilized to a solid support and theregion of interest is detected using miRNA sequence-specific ligationprobes as illustrated in FIG. 94, step D. In this embodiment, the miRNAsequence-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 94, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 94, step E).

Another aspect of the present invention is directed to a method foridentifying, in a sample, one or more micro-ribonucleic acid (miRNA)molecules containing a target micro-ribonucleotide sequence differing insequence from other miRNA molecules in the sample by one or more bases.This method involves providing a sample containing one or more miRNAmolecules potentially containing a target micro-ribonucleotide sequencediffering in sequence from other miRNA molecules by one or more basedifferences, and contacting the sample with one or more enzymes capableof digesting dU containing nucleic acid molecules potentially present inthe sample. The contacted sample is blended with a ligase and a firstoligonucleotide probe comprising a 5′ phosphate, a 5′ stem-loop portion,an internal primer-specific portion within the loop region, a blockinggroup, and a 3′ nucleotide sequence that is complementary to a 3′portion of the miRNA molecule containing a target micro-ribonucleotidesequence to form a ligation reaction. The method further involvesligating the miRNA molecule containing a target micro-ribonucleotidesequence at its 3′ end to the 5′ phosphate of the first oligonucleotideprobe to generate a chimeric nucleic acid molecule comprising the miRNAmolecule containing a target micro-ribonucleotide sequence, if presentin the sample, appended to the first oligonucleotide probe. One or moreoligonucleotide primer sets are provided, each primer set comprising (a)a first oligonucleotide primer comprising a 3′ nucleotide sequence thatis complementary to a complement of the 5′ end of the miRNA moleculecontaining a target micro-ribonucleotide sequence, and a 5′primer-specific portion, (b) a second oligonucleotide primer comprisinga nucleotide sequence that is complementary to the internalprimer-specific portion of the first oligonucleotide probe, and (c) athird oligonucleotide primer comprising a nucleotide sequence that isthe same as the 5′ primer-specific portion of the first oligonucleotideprimer. The chimeric nucleic acid molecule is blended with the one ormore second oligonucleotide primers, a deoxynucleotide mix includingdUTP, and a reverse transcriptase to form a reverse transcriptionreaction mixture, wherein the one or more second oligonucleotide primersof a primer set hybridizes to the internal primer specific portion ofthe chimeric nucleic acid molecule, and extends at its 3′ end togenerate a complement of the chimeric nucleic acid molecule, if presentin the sample. The method further involves blending the reversetranscription reaction mixture with the first and third oligonucleotideprimers of a primer set to form a polymerase reaction mixture, andsubjecting the polymerase chain reaction mixture to one or morepolymerase chain reaction cycles comprising a denaturation treatment, ahybridization treatment, and an extension treatment thereby formingprimary extension products. The primary extension products comprise the5′ primer-specific portion, a nucleotide sequence corresponding to thetarget micro-ribonucleotide sequence of the miRNA molecule, and theinternal primer-specific portion or complements thereof. The primaryextension products are blended with a ligase and one or moreoligonucleotide probe sets to form a ligation reaction mixture. Eacholigonucleotide probe set comprises (a) a first oligonucleotide probehaving a target-specific portion, and (b) a second oligonucleotide probehaving a target specific portion and a portion complementary to aprimary extension product, wherein the first and second oligonucleotideprobes of a probe set are configured to hybridize, in a base specificmanner, adjacent to one another on complementary target-specificportions of a primary extension product with a junction between them.The first and second oligonucleotide probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligation reaction mixture, and the ligated productsequences in the sample are detected and distinguished therebyidentifying one or more miRNA molecules containing a targetmicro-ribonucleotide sequence differing in sequence from other miRNAmolecules in the sample by one or more bases.

FIG. 95 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction to quantify miRNA. This method involves isolating RNA fromexosomes and treating with UDG for carryover prevention (FIG. 95, stepB). An oligonucleotide probe having a portion that is complementary tothe 3′ end of the target miRNA, and containing a stem-loop, tag (Tj′),and blocking group (filled circle) is ligated at its 5′ end to the 3′end of the target miRNA. The ligation product comprises the miRNA, Tj′tag, the blocking group, and a sequence complementary to the 3′ portionof the miRNA (FIG. 95, step B). cDNA is generated using a primer to Tj′and reverse transcriptase. The cDNA is amplified by Taq polymerase in alimited cycle PCR amplification (12-20 cycles) using a bridge primercomprising a sequence that is complementary to a portion of the reversetranscribed miRNA and an upstream primer-specific sequence portion (Ti)and tag (Ti, Tj) primers as shown in FIG. 95, step B. Alternatively, thecDNA is amplified using 20-40 PCR cycles. Primers contain identical 8-11base tails to prevent primer dimers. Optionally, the sample can bealiquot into 12, 24, 48, or 96 wells prior to PCR. PCR productsincorporate dU, allowing for carryover prevention (FIG. 95, step C).

As shown in FIG. 95, step D miRNA sequence-specific ligation probescontaining primer-specific portions (Ai, Ci′) suitable for subsequentPCR amplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 95, step D), and ligation products are aliquot intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probe (F1-Q) which spans the ligation junction (FIG. 95, step E-F). Thepresence of the miRNA in the sample is quantified using real-time PCRbased on the detection of the liberated label of the TaqMan™ probe.Samples are treated with UDG for carryover prevention, which alsodestroys original target amplicons (FIG. 95, step E). Only authentic LDRproducts will amplify, when using PCR in presence of dUTP. Neitheroriginal PCR primers nor LDR probes amplify LDR products, providingadditional carryover protection.

FIG. 96 illustrates another Ligation-RT-PCR-LDR-qPCR carryoverprevention reaction to quantify miRNA. This method involves essentiallythe same steps (i.e., steps A-D) as the method illustrated in FIG. 95;however, in this embodiment, the miRNA specific ligation probes aredesigned to contain UniTaq primer sequences (Ai, Ci′) and a UniTaq tagsequence (Bi′). Accordingly, in this embodiment, the ligation productsare subsequently amplified, detected, and quantified using real time PCRwith UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above andillustrated in FIG. 96, steps E-G.

FIG. 97 illustrates Ligation-RT-PCR-qLDR carryover prevention reactionto quantify miRNA. This method involves essentially the same steps,i.e., steps A and B, as described and illustrated in FIG. 95. However,in this embodiment, the cDNA that is produced in step B is amplifiedusing at least one biotin labeled primer to append a 5′ biotin to theamplification products containing the region of interest. PCR productsincorporate dU, allowing for carryover prevention (FIG. 97, step C). Thebiotinylated PCR products are immobilized to a solid support and theregion of interest is detected using miRNA sequence-specific ligationprobes as illustrated in FIG. 97, step D. In this embodiment, the miRNAsequence-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 97, step D), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 97, step E).

FIG. 98 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction to quantify miRNA. This method involves isolating RNA fromexosomes or another biological sample, and treating with UDG forcarryover prevention (FIG. 98, step B). An oligonucleotide probe havinga portion that is complementary to the 3′ end of the target miRNA, andcontaining a stem-loop, tag (Tj'), and blocking group (filled circle) isligated at its 5′ end to the 3′ end of the target miRNA. The ligationproduct comprises the miRNA, Tj′ tag, the blocking group, and a sequencecomplementary to the 3′ portion of the miRNA (FIG. 98, step B). cDNA isgenerated using primer Tj and reverse transcriptase. The cDNA isamplified by Taq polymerase in a limited cycle PCR amplification (12-20cycles) using a bridge primer comprising a sequence that iscomplementary to a portion of the reverse transcribed miRNA and anupstream primer-specific sequence portion (Ti) and tag (Ti, Tj) primers,where the bridge primer contains a cleavable blocking group on its 3′end as shown in FIG. 98, step C. Alternatively, the cDNA is amplifiedusing 20-40 PCR cycles. As described supra, C3-spacer is a suitableblocking group, and an RNA base (r) is cleaved using RNaseH (starsymbol) only when the primer hybridizes to its complementary target.Primers contain identical 8-11 base tails to prevent primer dimers.Optionally, the sample can be aliquot into 24, 48, or 96 wells prior toPCR. PCR products incorporate dU, allowing for carryover prevention(FIG. 98, step D).

As shown in FIG. 98, step E, miRNA sequence-specific ligation probescontaining primer-specific portions (Ai, Ci′) suitable for subsequentPCR amplification, hybridize to their corresponding target sequence in abase-specific manner. Ligase covalently seals the two oligonucleotidestogether (FIG. 98, step E), and ligation products are aliquot intoseparate wells for detection using tag-primers (Ai, Ci) and TaqMan™probe (F1-Q) which spans the ligation junction (FIG. 98, step F-G). Thepresence of the miRNA in the sample is quantified using real-time PCRbased on the detection of the liberated label of the TaqMan™ probe.Samples are treated with UDG for carryover prevention, which alsodestroys original target amplicons (FIG. 98, step F). Only authentic LDRproducts will amplify, when using PCR in presence of dUTP. Neitheroriginal PCR primers nor LDR probes amplify LDR products, providingadditional carryover protection.

FIG. 99 illustrates another Ligation-RT-PCR-LDR-qPCR carryoverprevention reaction to quantify miRNA. This method involves essentiallythe same steps (i.e., steps A-E) as the method illustrated in FIG. 98;however, in this embodiment, the miRNA specific ligation probes aredesigned to contain UniTaq primer sequences (Ai, Ci′) and a UniTaq tagsequence (Bi′). Accordingly, in this embodiment, the ligation productsare subsequently amplified, detected, and quantified using real time PCRwith UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above andillustrated in FIG. 99, steps F-H.

FIG. 100 illustrates Ligation-RT-PCR-LDR-qPCR carryover preventionreaction to quantify miRNA. This method involves essentially the samesteps, i.e., steps A and B, as described and illustrated in FIG. 98.However, in this embodiment, the cDNA that is produced in step B isamplified using at least one biotin labeled primer to form biotinylatedproducts containing the region of interest. PCR products incorporate dU,allowing for carryover prevention (FIG. 100, step D). The biotinylatedPCR products are immobilized to a solid support and the region ofinterest is detected using miRNA sequence-specific ligation probes asillustrated in FIG. 100, step E. In this embodiment, the miRNAsequence-specific ligation probes of a ligation pair containcomplementary tail sequences and an acceptor or donor group,respectively, capable of generating a detectable signal via FRET whenbrought in close proximity to each other as described supra.Accordingly, following ligation (FIG. 100, step E), the complementary 5′and 3′ tail ends of the ligation products hybridize to each otherbringing their respective donor and acceptor moieties in close proximityto each other to generate a detectable FRET signal (FIG. 100, step F).

As described in more detail herein, the method of the present inventionare capable of detecting low abundance nucleic acid molecules comprisingone or more nucleotide base mutations, insertions, deletions,translocations, splice variants, miRNA variants, alternativetranscripts, alternative start sites, alternative coding sequences,alternative non-coding sequences, alternative splicings, exoninsertions, exon deletions, intron insertions, other rearrangement atthe genome level, and/or methylated nucleotide bases.

As used herein “low abundance nucleic acid molecule” refers to a targetnucleic acid molecule that is present at levels as low as 1% to 0.01% ofthe sample. In other words, a low abundance nucleic acid molecule withone or more nucleotide base mutations, insertions, deletions,translocations, splice variants, miRNA variants, alternativetranscripts, alternative start sites, alternative coding sequences,alternative non-coding sequences, alternative splicings, exoninsertions, exon deletions, intron insertions, other rearrangement atthe genome level, and/or methylated nucleotide bases can bedistinguished from a 100 to 10,000-fold excess of nucleic acid moleculesin the sample (i.e., high abundance nucleic acid molecules) having asimilar nucleotide sequence as the low abundance nucleic acid moleculesbut without the one or more nucleotide base mutations, insertions,deletions, translocations, splice variants, miRNA variants, alternativetranscripts, alternative start sites, alternative coding sequences,alternative non-coding sequences, alternative splicings, exoninsertions, exon deletions, intron insertions, other rearrangement atthe genome level, and/or methylated nucleotide bases.

In some embodiments of the present invention, the copy number of one ormore low abundance target nucleotide sequences are quantified relativeto the copy number of high abundance nucleic acid molecules in thesample having a similar nucleotide sequence as the low abundance nucleicacid molecules. In other embodiments of the present invention, the oneor more target nucleotide sequences are quantified relative to othernucleotide sequences in the sample. In other embodiments of the presentinvention, the relative copy number of one or more target nucleotidesequences is quantified. Methods of relative and absolute (i.e., copynumber) quantitation are well known in the art.

The low abundance target nucleic acid molecules to be detected can bepresent in any biological sample, including, without limitation, tissue,cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodilyfluids, bodily secretions, bodily excretions, cell-free circulatingnucleic acids, cell-free circulating tumor nucleic acids, cell-freecirculating fetal nucleic acids in pregnant woman, circulating tumorcells, tumor, tumor biopsy, and exosomes.

The methods of the present invention are suitable for diagnosing orprognosing a disease state and/or distinguishing a genotype or diseasepredisposition.

With regard to early cancer detection, the methods of the presentinvention are suitable for detecting both repeat mutations in knowngenes (e.g., CRAF, KRAS), and uncommon mutations in known genes (e.g.,p53) when present at 1% to 0.01% of the sample. The methods of thepresent invention can also achieve accurate quantification oftumor-specific mRNA isolated from exosomes (e.g. a dozen expressionmarkers that differentiate colon tumor tissue from matched normalmucosa), and tumor-specific miRNA isolated from exosomes or Argonautproteins (e.g. a dozen microRNA markers that differentiate colon tumortissue from matched normal mucosa). The methods of the present inventionalso afford accurate quantification of tumor-specific copy changes inDNA isolated from circulating tumor cells (e.g. a dozen copy changesthat differentiate colon tumor tissue from matched normal mucosa), andthe detection of mutations in DNA isolated from circulating tumor cells.(e.g. KRAS, BRAF, AKT, p53, BRCA1 genes).

The present invention is also capable of accurately quantifying (i)tumor-specific mRNA isolated from exosomes or circulating tumor cells,(ii) tumor-specific miRNA isolated from exosomes or Argonaut proteins,and (iii) tumor-specific copy changes in DNA isolated from circulatingtumor cells that can predict outcome or guide treatment. The presentinvention can also detect mutations in DNA isolated from circulatingtumor cells, e.g. KRAS, BRAF, AKT, p53, BRCA1 or other genes thatpredict outcome or guide treatment.

With regard to prenatal diagnostics, the methods of the presentinvention are capable of detecting aneuploidy through counting copynumber (e.g., Trisomy 21), inherited diseases containing commonmutations in known genes (e.g. Sickle Cell Anemia, Cystic Fibrosis),inherited diseases containing uncommon mutations in known genes (e.g.familial adenomatous polyposis), inherited diseases arising from knownor sporadic copy number loss or gain in known gene (e.g. Duchenne'smuscular dystrophy), and paternity testing.

An important aspect of implementing the assays described above in aclinical setting is the reduction in the amount of labor required tofill rows and columns with samples, reagents and assay specificprobes/primers. Ninety-six well plates are the standard in mostlaboratories, but 384 well plates afford a higher throughput andreduction in the cost of each assay well. Part of the hindrance of theiradoption has been the increased labor associated with these plates,which can be solved by pipetting robots but at considerable capitalexpense. Depending upon the specific configuration of assays in theplate, significant expense is also incurred by the increased use ofpipette tips. One embodiment of the assays described above requires thedispersal of 24 multiplex PCR-LDR reactions into the 24 columns of amicrotiter plate followed by the dispersal of 16 different sets of LDRtag probes across the rows of the plate. This assay set-up would require48 deliveries by an 8 tip pipettor to fill the columns and then 48deliveries by an 8 tip pipettor to fill all of the rows. Plates having1536 wells have the advantage of reducing the cost of an assay evenfurther but demand automated filling as they are beyond the mechanicalabilities of a human operator. Devices have been commercialized thatallow the simultaneous filling of many rows and columns with a reducednumber of pipetting steps by the use of microfluidic devices that uselow dead volume channels that introduce liquids into each “well” butthat require the added complication of valves and external valvedrivers. Clearly a different approach is warranted.

Another aspect of the present invention is directed to a device for usein combination with a microtiter plate to simultaneously add liquids totwo or more wells in a row and/or column of the microtiter plate havingopposed top and bottom surfaces with the top surface having openingsleading into the wells and the bottom surface defining closed ends ofthe wells. The device comprises a first layer defined by first andsecond boundaries with metering chambers extending between the first andsecond boundaries of said first layer and in fluid communication withone another. The first layer is configured to be fitted, in an operativeposition, proximate to the microtiter plate with the first boundary ofthe first layer being closest to the top surface of the microtiter plateand each of the metering chambers being in fluid communication with anindividual well in a row and/or column of the microtiter plate. Thefirst layer further comprises a filling chamber in fluid communicationwith one or more of the metering chambers. The device comprises a secondlayer defined by first and second boundaries with a filling portextending between the first and second boundaries of the second layer.The second layer is configured to be fitted, in an operative position,proximate to the first layer with the first boundary of the second layerbeing closest to the second boundary of the first layer and the fillingport being aligned with the filling chamber. When the first layer,second layer, and microtiter plate are positioned with respect to oneanother in their operative positions, liquid entering the device throughthe filling port will pass through the filling chamber, the meteringchambers, and into two or more wells in a row and/or column of themicrotiter plate.

In some embodiments, the device of the present invention furthercomprises an intermediate layer having first and second boundaries withintermediate layer passages extending between the first and secondboundaries of said intermediate layer. The intermediate layer isconfigured to be fitted, in an operative position, between themicrotiter plate and the first layer with the first boundary of theintermediate layer adjacent to the top surface of the microtiter plateand the second boundary of the intermediate layer adjacent to the firstboundary of the first layer. One of the intermediate layer passages isaligned with an individual well in a row and/or column of the microtiterplate, where, when the first layer, the second layer, the intermediatelayer, and the microtiter plate are positioned with respect to oneanother in their operative positions, liquid entering the device throughthe filling port will pass through the filling chamber, the meteringchambers, the intermediate layer passages, and into the wells of themicrotiter plate.

The device of the present invention may further comprise a third layerhaving first and second boundaries with a filling port connectorextending between the first and second boundaries of the third layer.The third layer is configured to be fitted, in an operative position,between the first and the second layers with the first boundary of thethird layer adjacent to the second boundary of the first layer and thefilling port connector being aligned with the filling port. When thefirst layer, the second layer, the third layer, the intermediate layer,and the microtiter plate are positioned with respect to one another intheir operative positions, liquid entering the device through thefilling port will pass through the filling port connector, the fillingchamber, the metering chambers, the intermediate layer passages, andinto the wells of the microtiter plate.

FIGS. 103-112 depict one exemplary embodiment of the device of theinvention. FIG. 103 shows a top and side view of a portion of a typical384-well microtiter plate 100 that is used in combination with thedevice of the present invention. The microtiter plate is defined byseveral wells 103, each well having a top, open end 104 that is suitablefor receiving sample and/or reaction reagents, and a closed, bottom end102. FIG. 104 shows a perspective view of the microtiter plate of FIG.103.

FIGS. 105 and 106 depict top and side views and an exploded perspectiveview, respectively, of the intermediate layer 106 of the devicepositioned adjacent to the top surface of the microtiter plate 100. Theintermediate layer 106 of the device contains intermediate passages 108that extend through the intermediate layer. Each intermediate passage108 of the intermediate layer 106 aligns with an individual well 103 ofa row or column of the microtiter plate 100. In one embodiment, theintermediate layer passages function as burst valves to control orprevent the flow of liquid from the metering chamber into the wells ofthe microtiter plate. The diameter of the vertical channel of theintermediate layer passages 108 creates surface tension that preventsliquid from flowing out of the metering chamber 120 (see FIG. 107) untilcentrifugal force is applied. In some embodiments, the vertical walls ofthe intermediate layer passages 108 are composed of or are coated with ahydrophobic material which increases the resistance of fluid flow out ofthe metering chamber 120 until centrifugal force is applied. Suitablehydrophobic materials include any material with a water contactangle >90°, such as, e.g., cyclic olefin copolymer, polyethylene,polypropylene, polydimethylsiloxane, fluorinated ethylene polypropylene,polytetrafluoroethylene. Alternatively, the wall of the intermediatelayer passages may be composed of a hydrophilic material having a watercontact angle <90°, but treated with a hydrophobic coatings, e.g.,Teflon-carbon black, to create a superhydrophobic surface having watercontact angles >150°.

The ratio of the resistance of the intermediate layer passages 108 tothe volume of the metering chamber 120 (see FIG. 107) is readilycalculable by one skilled in the art. Other embodiments of passivevalves that release the liquid under the influence of an externallyapplied force can alternatively be employed.

As depicted in FIGS. 105 and 106, the intermediate layer 106 alsocontains an overflow passage 110 that connects the overflow chamber 116of the first layer with the filling chamber 118 of the first layer (seeFIG. 107).

Proper positioning and orientation of the device onto the top surface ofa microtiter plate is achieved by keying the device to the perimeter ofthe top of the microtiter plates as shown in FIGS. 101 and 102. Furtheralignment of each of the metering chambers can be achieved by the use offlanges or skirts 112 in the intermediate layer 106 which interface withthe open end 104 of each well 103 of the microtiter plate 100 as shownin the side view of FIG. 105. Other embodiments of positioning can beenvisioned by one skilled in the art, for example based on small flangesat the top of the wells of the microtiter plate. While not meant toprovide a hermetic seal, the flanges or skirts 112 of the intermediatelayer 106 as depicted in FIG. 105 provide some measure of crosscontamination control between adjacent wells of the microtiter plate100.

FIGS. 107 and 108 depict top and side views and an exploded perspectiveview, respectively, of the first layer 114 of the device, operativelypositioned adjacent to the second boundary of the intermediate layer 106of the device. The first layer 114 of the device contains meteringchambers 120 that are in fluid communication with each other viametering chamber channels 122, and with individual wells 103 of themicrotiter plate. The metering chambers 120 have a fixed volume tocontrol the volume of liquid delivered into each well 103 of themicrotiter plate 100. The metering chambers 120 receive liquid from thefilling chamber 118 by capillary action of the liquid or by mechanicalforce, e.g., the force of a pipettor pushing liquid into the fillingchamber 118. In one embodiment, the metering chambers 120 of the deviceall have the same metering volume. In another embodiment, the meteringchambers 120 have differing metering volumes per row and/or column. Thewalls of the filling chamber, metering chambers, and the meteringchamber channels may be composed of or coated with a hydrophilicmaterial.

As illustrated in FIGS. 107 and 108, each metering chamber 120 is influid communication with the wells 103 of the microtiter plate via theintermediate layer passages 108. As described supra, the intermediatelayer passages may function as a burst valve to prevent liquid in themetering chamber 120 from flowing into the wells 103 of the microtiterplate 100 until appropriate force is applied, e.g., centrifugal force.

The first layer 114 of the device also contains an overflow chamber 116as depicted in FIGS. 107 and 108. As noted above, the overflow chamber116 is in fluid communication with the filling chamber 118 via theoverflow passage 110 of the intermediate layer 106.

FIGS. 109 and 110 depict top and side views and an exploded perspectiveview, respectively, of the third layer 124 of the device operativelypositioned adjacent to the second boundary of the first layer 114 of thedevice. The third layer 124 of the device contains a filling portconnector 128 that extends through the third layer 124, aligning andconnecting the filling port 132 of the second layer 130 (shown in FIG.111) with the filling chamber 118 of the first layer 114. The thirdlayer 124 also contains air passage connectors 126 that extend throughthe third layer 124, aligning with and connecting the metering chambers120 of the first layer 114 with the air passages 136 of the second layer130 (also shown in FIG. 111). The walls of the air passage connectors126 of the third layer are composed of or coated with a hydrophobicmaterial. The third layer 124 of the device also contains overflow airpassage connectors 125 that extend through the third layer, aligningwith and connecting the overflow chamber 116 of the first layer 114 tothe overflow air passages 134 of the second layer 130 (shown in FIG.111).

FIGS. 111 and 112 depict top and side views and an exploded perspectiveview, respectively, of the second layer 130 of the device operativelypositioned adjacent to the second boundary of the third layer 124 of thedevice. The second layer 130 of the device contains a filling port 132that extends through the second layer 130, and aligns with the fillingport connector 128 of the third layer 124, or in some embodiments,directly with the filling chamber 118 of the first layer 114. The secondlayer 130 of the device also contains air passages 136 that extendthrough the second layer 130 and align with the metering chambers 120 ofthe first layer 114. The air passages 136 of the second layer connect tothe metering chambers 120 of the first layer 114 via the air passageconnectors 126 of the third layer 124. As further illustrated in FIGS.111 and 112, the second layer 130 of the device also contains anoverflow air passage 134 that extends through the second layer 130 andaligns with the overflow chamber 116 of the first layer 114. Theoverflow air passage 134 connects to the overflow chamber 116 via theoverflow air passage connectors 125 of the third layer 124.

Although the device is described in terms of individual layers, thelayers of the device are integral, giving the device a monolithicstructure.

In one embodiment of the present invention the first layer of the deviceis provided with a pair of spaced filling chambers on opposite ends ofthe metering chambers and the second layer is provided with a pair ofspaced filling ports, each in fluid communication with one of the pairof spaced filling chambers. One of the pair of spaced filling portsprovides liquid to one of the pair of spaced filling chambers and halfof the metering chambers, while the other one of the pair of spacedfilling ports provides liquid to the other of the pair of fillingchambers and the other half of the metering chambers.

In another embodiment of the present invention, the first layer of thedevice is provided with a pair of spaced filling chambers on oppositeends of the metering chambers and the second layer is provided with apair of spaced filling ports, each in fluid communication with one ofthe pair of spaced filling chambers. One of the pair of spaced fillingports provides liquid to one of the pair of filling chambers and all ofthe metering chambers, while the other one of the pair of spaced fillingports provides liquid to the other of the pair of filling chambers andall of the metering chambers.

The device of the present invention can be configured to fill two ormore rows and columns of said microtiter plate with liquid.

The Figures herein illustrate different designs compatible with a 384well microtiter plate; however the concepts described are likewiseapplicable to 1536 well plates by one skilled in the art. FIG. 105 thru112 described in detail above illustrate a device for the simultaneousfilling of all wells in all rows across 24 columns by use of fillingports 132 on the right side of the device-microtiter plate stack. Thisconfiguration relies on capillary action to fill each of the individualmetering chambers 120 connected by channels 122 as shown in FIG. 111.

FIGS. 113 thru 118 are a series of top, side, and exploded perspectivedrawings that illustrate a second configuration of the device. Thissecond configuration of the device relies on mechanical force of thepipettor to drive the liquid into the channels and metering chambers.FIGS. 113-118 depict all of the same device features as shown in FIGS.107-112 (numbered correspondingly as 200-236), except that the fillingport 232 of the second layer 230 is modified to facilitate mechanicalfilling of the wells with liquid as shown in FIG. 117. The filling port232 dimensions are tapered, such that a standard disposable tip wouldfit snugly into the port, allowing for positive pressure to be used tofill the metering chambers. The dimensions and spacing of the fillingports are compatible with use of hand-held multichannel pipettes ormultichannel robotic workstations.

FIGS. 119 thru 126 are a series of top, side, and exploded perspectivedrawings that show a different portion of the device as depicted inFIGS. 105 thru 118. The portion of the device depicted in FIGS. 119-126represents the exit ends of the row and/or columns shown in FIGS.105-118, i.e., the portion of the device that is opposite the fillingports. The device features depicted in FIGS. 119-126, labeled as300-336, correspond to the features described and labeled in referenceto FIGS. 105-112 (i.e., device features 100-136).

FIGS. 127 thru 134 are a series of top, side, and exploded perspectivedrawings that illustrate an alternative configuration of the device ofthe present invention. In this configuration of the device, fillingports 432 (see FIG. 133) for loading reagents into the wells of themicrotiter plate are located on both sides of the device-microtiterplate stack. This configuration can be used to add two different sets ofreagents to all wells in each row. Alternatively, each side of thefluidic channels can address 12 of the 24 columns from each side of thedevice thus dividing the plate into two side-by-side regions of 192wells. This configuration retains the ability to independently addresseach row and each column of the two regions. The device featuresdepicted in FIGS. 127-134, labeled as 400-436, correspond to thefeatures described and labeled in reference to FIGS. 105-112 (i.e.,device features 100-136).

FIGS. 135 thru 144 are a series of top, side, and exploded perspectivedrawings that illustrate an alternative configuration of the device ofthe present invention. This configuration of the device is suitable forthe simultaneous filling of all wells by rows and all wells by columns.Alternatively, each side of the fluidic channels can address 12 of the24 columns from each side of the device and 8 of the 16 rows from eachside of the device thus dividing the plate into four different regionsof 96 wells. This configuration retains the ability to independentlyaddress each row and each column of the four regions. The devicefeatures depicted in FIGS. 135-144, labeled as 500-536, correspond tothe features described and labeled in reference to FIGS. 105-112 (i.e.,device features 100-136).

In accordance with this configuration, FIGS. 135 and 136 depict top andside views and an exploded perspective view, respectively, of theintermediate layer 506 of the device positioned adjacent to the topsurface of the microtiter plate 500. The intermediate layer 506 of thedevice contains intermediate passages 508 that extend through theintermediate layer. In this embodiment, each well of the microtiterplate aligns with at least two intermediate passages 508 as shown in thetop, side and exploded perspective view of FIGS. 135 and 136,respectively. The intermediate layer 506 also contains the overflowpassage 510.

FIGS. 137 thru 140 depict top, side, and an exploded perspective views,of the first layer 514 of the device (see FIG. 139), operativelypositioned adjacent to the second boundary of the intermediate layer 506of the device. In accordance with this embodiment, the first layer 514of the device of the present invention has a first region 513 having themetering chambers 520 in two or more rows (see FIGS. 137 and 138), and asecond region 515 having the metering chambers 520 in two or morecolumns (see FIGS. 139 and 140) with the first and second regions beingdisplaced from each other. The metering chambers 520 of the first 513and second 515 regions of the first layer 514 are in fluid communicationwith each other via metering chamber channels 522, and with individualwells 503 of the microtiter plate. As described supra, the meteringchambers 520 have a fixed volume to control the volume of liquiddelivered into each well 503 of the microtiter plate 500. The meteringchambers 520 receive liquid from the filling chamber 518 by capillaryaction of the liquid or by mechanical force pushing liquid into thefiling chamber 518. Flow of the liquid out of the metering chambers 520into the wells of the microtiter plate is controlled by, e.g., thehydrophobic forces of the intermediate layer passages 508. In oneembodiment, the metering chambers 520 of the device all have the samemetering volume. In another embodiment, the metering chambers 520 havediffering metering volumes per row and/or column.

The first 513 and second 515 regions of the first layer 514 of thedevice each contain an overflow chamber 516 as depicted in FIGS. 137-138and FIGS. 139-140, respectively. As noted above, the overflow chamber516 is in fluid communication with the filling chamber 518 via theoverflow passage 510 of the intermediate layer 506.

FIGS. 141 and 142 depict top and side views and an exploded perspectiveview, respectively, of the third layer 524 of the device operativelypositioned adjacent to the second boundary of the second region 515 ofthe first layer 514 of the device. The third layer 524 of the devicecontains a filling port connector 528 that extends through the thirdlayer 524, aligning and connecting the filling port 532 of the secondlayer 530 (shown in FIG. 143) with the filling chamber 518 of the firstlayer 514. The third layer 524 also contains air passage connectors 526that extend through the third layer 524, aligning with and connectingthe metering chambers 520 of the first layer 514 with the air passages536 of the second layer 530 (also shown in FIG. 143). The third layer524 of the device also contains overflow air passage connectors 525 thatextend through the third layer, aligning with and connecting theoverflow chamber 516 of the first layer 514 to the overflow air passages534 of the second layer 530 (shown in FIG. 143).

FIGS. 143 and 144 depict top and side views and an exploded perspectiveview, respectively, of the second layer 530 of the device operativelypositioned adjacent to the second boundary of the third layer 524 of thedevice. In this embodiment, the second layer 530 of the device containsfilling ports 532, one for each row and each column, that extend throughthe second layer 530, and align with the filling port connectors 528 ofthe third layer 524, or in some embodiments, directly with the fillingchambers 518 of the first layer 514. The second layer 530 of the devicefurther contains air passages 536 that extend through the second layer530 and align with the metering chambers 520 of the first layer 514. Theair passages 536 of the second layer connect to the metering chambers520 of the first layer 514 via the air passage connectors 526 of thethird layer 524. As further illustrated in FIGS. 143 and 144, the secondlayer 530 of the device also contains an overflow air passage 534 ineach row and column that extends through the second layer 530 and alignswith the overflow chamber 516 of the first layer 514. The overflow airpassage 534 connects to the overflow chamber 516 via the overflow airpassage connectors 525 of the third layer 524.

While each of the different configurations described above isillustrated by a series of drawings that show top, side, and explodedviews of each layer of the device, for purposes of fabrication, thedevice can be molded monolithically or assembled from individual layersas determined by one skilled in the art.

Another aspect of the present invention is directed to a method ofadding liquids to two or more wells in a row and/or column of amicrotiter plate having opposed top and bottom surfaces with the topsurface having openings leading into the wells and the bottom surfacedefining closed ends of the wells. This method involves providing thedevice of the present invention as described supra, and filling thedevice with liquid. The liquid is discharged into two or more wells in arow and/or column of said microtiter plate of the device.

As noted above, the device of the present invention may be configured topermit the wells to be filled by capillary action or by mechanicalforce.

The device of the present invention, which allows the simultaneousfilling of all columns and all rows of a microtiter plate eithersequentially or at the same time, is fabricated out of a suitablematerial (e.g., polystyrene, polycarbonate, etc.) that is compatiblewith biological reagents and is positioned over wells of the microtiterplate. Next, reagents are introduced into the filling ports (e.g., 24and/or 16 filling ports) and the reagents are automatically dispersed toeach of the metering chambers positioned above each of the microtiterplate wells (e.g., 384 metering chambers for 384 wells in a plate). Theloaded device-microtiter plate stack is placed into a swinging bucketrotor of a standard low speed centrifuge and subjected to a brief spinat a force sufficient to drive the liquid from the individual meteringchambers into each of the wells. After the centrifuge has halted, thedevice-microtiter plate stack is removed from the centrifuge, the stackis separated and the device is disposed of while the plate is then usedfor the next step in the assay process. Thus the labor set-up of theassay configuration described above for a 384 well plate would bereduced to 3 each 8-tip pipettor deliveries to load the column fillingports and 2 each 8-tip pipettor deliveries to load the row filling portscompared to the 96 total 8-tip pipettor deliveries using a manualapproach. In addition, the use of the device consumes only 24 pipettetips while a manual approach would consume 384 pipette tips for aconsiderable cost savings, which is important in a high throughputclinical laboratory. The device and process described above could easilybe applied to a 1536 well microtiter plate by one skilled in the art andwould result in similar benefits as described for the 384 well plate. Anadded benefit of the device described herein, which dramatically reducesthe number of pipetting steps, is the control of cross contamination byaerosols containing PCR amplicons. This is especially critical in thedetection of low copy number representation of mutant alleles. Duringthe introduction of liquids to the loading ports of the device, each ofthe 384 or 1536 wells is covered by the device and the combination ofthe covered wells and the reduction in the number of liquid transfersteps provides some decreased probability of PCR amplicon crosscontamination between wells.

Since each row and column is independently addressable, one can conceiveof many assay configurations that can be fulfilled by the same device bythe judicious choice of how the loading ports are filled. Thus the sameliquid can be applied to all 24 columns by 3 each 8-tip pipetting steps(no tip changes) and the same liquid can be applied to all 16 rows by 2each 8-tip pipetting steps (no tip changes); 24 different components canbe applied to each of the 24 rows by 3 each 8-tip pipetting steps (3 tipchanges) and 16 different components can be applied to each of the 16rows by 2 each 8-tip pipetting steps (2 tip changes). Many other fillingconfigurations are possible to one skilled in the art. The dispersedliquid can be any biological liquid, e.g., a biological sample, reactionreagents such as, e.g., primers, probes, enzymes, reaction products,etc.

In one embodiment, a series of dispersion devices are available with achoice of fixed metering volumes, which anticipates particular highvolume molecular biology applications such as might be found in aclinical diagnostics laboratory or a pharmaceutical developmentlaboratory. In another embodiment, custom dispersion devices are madewith user specified metering volumes for their specific applications.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

EXAMPLES Prophetic Example 1 High Sensitivity Mutation Marker (WhenPresent at 1% to 0.01%), Single Base Mutation, Small Insertion, andSmall Deletion Mutations in Known Genes in Total Plasma cfDNA

Overview of approach: This approach depends on the fidelity of threeenzymes: (i) Taq polymerase to faithfully copy low-level copies of DNAin the initial sample, (ii) RNase H2 enzyme removing a blocking group onthe upstream LDR primer, and (iii) Ligase in discriminating a match frommismatch on the 3′ side of the upstream primer. The later is enhancedfurther by using an intentional mismatch or nucleotide analogue in the2^(nd) or 3^(rd) base from the 3′ end that slightly destabilizeshybridization of the 3′ end if it is perfectly matched at the 3′ end,but significantly destabilizes hybridization of the 3′ end if it ismis-matched at the 3′ end. Finally, kinetic approaches, such as alteringthe cycling times and conditions can enhance the discrimination betweenwild-type and mutant template. Once a ligation event has taken place,those products will be amplified in a subsequent real-time PCRamplification step, and thus this is the key discriminatory step.

For the initial PCR step, PCR primers containing universal tails thatare partially identical are used at lower concentrations (10-50 nM). Theidentical region may vary from 8 to 11 bases or more. Thus, if anytarget-independent primer dimer formed, the incorrect product will forma hairpin that will inhibit further amplification. Further, the tailsenhance subsequent binding of PCR primer to the correct amplicons.

Alternatively, the fidelity of amplification and reduction of falseamplicons and primer dimers is achieved by using PCR primers at lowerconcentrations, but containing an RNA base, 4 additional bases and ablocking group on the 3′ end. Only when the primer correctly hybridizesto its intended target will RNaseH2 cleave the RNA base, liberating afree 3′OH on the DNA primer. Even if a primer is accidentally activatedon an incorrect target, on the next round, the bases downstream of theprimer 3′ end will not be perfect matches for the bases to be removed.This significantly lowers the chance of a second cleavage and extension.In short the efficiency of amplification of incorrect targets will notproduce significant background. Further, the initial PCR amplificationis followed by an LDR step, essentially nesting within the initial PCRproduct.

When starting with cfDNA, the average length is 160 bases, thus PCRprimers should be pooled in two groups when tiling across a gene region(e.g., p53) such that each group amplifies fragments of about 100 bp,which are shifted with respect to each other by about 50 bases, suchthat one gets “tiling” across a given region.

To protect against carryover contamination, UNG is added to the reactionprior to polymerase activation, and the initial PCR amplification isperformed with dUTP. The LDR probes are comprised of the natural bases,thus the LDR product is now resistant to UNG digestion in the secondreal-time PCR step. Note that the LDR products contain sequence tags orUniTaq sequences on their non-ligating ends, which are lacking in thetarget DNA, thus accidental carryover of LDR products does not result inlarge-scale amplification. Unlike with PCR, an initial LDR product isnot a substrate for a second LDR reaction.

The most difficult case is for KRAS mutations, where 6 changes on codon12 and 1 change on codon 13 are all spaced together. In general, forhighest fidelity, the mismatch between mutant probe and wild-typesequence should at least be C:A for the last base, not G:T. Thus, oneneeds to run both upper-strand and lower-strand probes, or 2 ligationsets per PCR reaction. However, more than one mutation may be given thesame UniTaq sequence, or fluorescence label with a TaqMan™ probe, sincethe aim is to find a mutation and not necessarily distinguish differentmutations from each other.

Since the different probes will compete with each other in binding the(rare) mutant sequence, it is important to allow for all the probes tohybridize to the correct sequence. There will be 3 upstream and 1downstream primers for the KRAS codon 12 1s^(t) position mutations.False ligation of mutant LDR probes on wild-type target sequence may befurther suppressed by using blocked upstream LDR probe with thewild-type sequence at the discriminating base, but lacking theappropriate tag sequence Probes are designed to avoid falseligation/false signal of mutant probes to normal sequence, but also forcorrect ligations to occur in the presence of the mutant sequence.

To summarize the levels of discrimination of the above approach usingboth PCR primers and LDR probes for detection of each mutation:

-   1. Use of PCR primers with universal tails, such that if any    target-independent primer dimer formed, the incorrect product will    form a hairpin that will inhibit further amplification.-   2. Use of UNG to prevent carryover contamination of initial PCR    reaction.-   3. Use of nuclease activity of RNaseH2 to liberate an unblocked 3′    OH on the upstream LDR probe, only when hybridized to target.-   4. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   5. Use of mismatch or nucleotide analogue in the 2^(nd) or 3^(rd)    base from the 3′ end of upstream probe.-   6. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   7. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed Protocol For Highly Sensitive Detection Of Mutation Marker    (when Present at 1% to 0.01%), repeat mutations in known genes:

1.1.a. Incubate genomic DNA in the presence of UNG (37° C., 15-30minutes, to prevent carryover), dUTP, and other dNTP's, AmpliTaq Gold,and gene-specific primers containing universal tails, such that if anytarget-independent primer dimer formed, the incorrect product will forma hairpin that will inhibit further amplification. This initial genomicDNA—PCR reaction mixture is suitable for multiplex PCR amplification in12, 24, 48, or 96 individual wells (spatial multiplexing), or in asingle well. Denature genomic DNA from plasma, inactivate UNG, andactivate AmpliTaq Gold (94° C., 5-10 minute) and multiplex PCR amplifymutation-containing fragments for a limited number of cycles (94° C., 10sec., 60° C. 30 sec., 72° C. 30 sec. for 12-20 cycles). The PCR primersare designed to have Tm values around 64-66° C., and will hybridizerobustly, even when used at concentrations 10 to 50-fold below the normfor uniplex PCR (10 nM to 50 nM each primer). The cycles are limited toretain proportional balance of PCR products with respect to each other,while still amplifying low abundant sequences about 100,000 to1,000,000—fold. After PCR amplification, Taq polymerase is inactivated(by incubating at 99° C. for 30 minutes.)

1.1.b. Add thermostable ligase (preferably from strain AK16D), RNaseH2,buffer supplement to optimized ligation conditions, and suitableupstream and downstream LDR probes (10 nM to 20 nM each, downstreamprobes may be synthesized with 5′ phosphate, or kinased in bulk prior toreactions; upstream probes comprise an RNA base after the desired 3′end, 4 additional bases, and a blocking group to preventtarget-independent ligation.) Upstream probes comprise of a 5′ tag, suchas UniAi followed by target-specific sequence with a C:A or G:T mismatchat the 3^(rd) or penultimate base, the mutation base at the 3′ end,followed by an RNA base and 4 more DNA bases that matches the target,and a C3 spacer to block ligation (or subsequent extension bypolymerase). The downstream probes comprise a 5′ phosphorylated end,followed by target-specific sequence, and a 3′ tag, such as UniCi′.Perform 20 cycles of LDR, (94° C., 10 sec., 60° C. 4-5 minutes). Thiswill allow for ligation events to occur on the PCR products if mutantDNA is present.

1.1.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR probes would be UniAi and UniCi respectively,and the products would be of the form:

UniAi—Upstream Target-Mutation-Downstream Target—UniCi′

This approach avoids generating background signal off wild-type DNA inthe second real-time PCR reaction. First, UNG will destroy the bottomstrand of the initial PCR product, such that remaining upstream LDRprobe has no target to hybridize to, and thus the 3′ end remains blockedand will not extend. Second, any residual PCR primer from the initialPCR reaction will be unable to bind to either initial PCR products(destroyed by UNG) or LDR products (no binding sites) and thus the 3′end remains blocked and will not extend. Finally, the TaqMan™ probe nowhas 2 bases differing from wild-type sequence (the mutation base, andthe base in the 3^(rd) position from the 3′ end of the ligationjunction), and thus will only hybridize at a temperature below 60° C.,but now the upstream PCR primer will have hybridized first, andconsequently extended, thus preventing the TaqMan™ probe fromhybridizing and generating signal from the 5′-3′ activity of the Taqpolymerase.

A second assay design is based on an initial multiplexed PCRamplification followed by distribution and capture of PCR amplifiedtargets on the wells of a microtiter plate. A single cycle of LDRenables capture of LDR products on the correct targets on the solidsupport, while mis-ligations are washed away. The LDR products arequantified, either through LDR-FRET, real-time PCR, or other reportersystems.

For amplifying cfDNA for mutation detection, PCR primers containinguniversal tails are used. The gene-specific primers are used at lowconcentrations (10 to 50 nM) and contain universal tails that arepartially identical. Thus, if there is any target-independent primerdimer formed, the product will form a hairpin that will inhibit furtheramplification. To maximize the ability to detect very low abundancemutations, after making a master mix containing all the components, thereaction mixture is distributed into 12, 24, 48, or 96 independentwells. Since a single molecule (with a mutation) can only be distributedinto a given well, the process will effectively enrich themutation-containing molecule compared to the normal wild-type DNA, andthus significantly improve signal-to-noise. One approach is to use atwo-step amplification, wherein the initial amplification usesgene-specific primers with universal tails, and the second amplificationuses universal primers to append a biotin group to a specific productstrand. The initial amplification (with low PCR primer concentration)will still be quantitative, provided it is limited to about 8-20 cycles.The amplification products are then diluted into two new wells for eachof the original wells, each containing the two universal primers (athigher concentrations of 0.5-1 pmoles), with one or the otherbiotinylated in the respective well. Amplification is now continued foranother 8-29 cycles, for a total of about 15-40 cycles. As an optionalstep, the products may be separated (by electrophoresis or size) fromunused primers.

Alternatively, products may be amplified in a single amplificationreaction by adding the universal primers to the initial amplificationwell, where only one universal primer is biotinylated. Alternatively,biotinylated gene-specific primers may be used directly in a singleamplification step. Only when it is prudent to design LDR probes againstboth the top and bottom strand (i.e. to maximize LDR discrimination ofmutation) will it be necessary to capture both forward and reversestrand of a given amplicon. This may be achieved by using mixes of eachprimer at 50% biotinylation in the same reaction, or at 100%biotinylation in separate reactions. As long as the biotinylatedproducts remain separated during the capture on the solid support, theymay both be in the same amplification reaction. However, if the PCRproduct strands rehybridize after capture, they may need to be capturedon separate addresses on the solid support. This spatial separation maybe needed to assure there is sufficient single-stranded PCR productavailable for identifying mutations by subsequent LDR detection.

With cfDNA, fragment length is biologically limited to about 160 bp.Thus, in order to cover common hot-spot mutations across a largerregion, primer sets will be designed to generate overlapping fragments.As such, the primers would be distributed between an “A” and “B” pool,doubling the number of wells mentioned above. With DNA isolated fromCTC's exon size fragments may often be used, thus mitigating the needfor two amplicon pools. Some fragments amplify more slowly than others.This problem may be overcome by including an additional multiplexedreaction with a few more amplification cycles.

FIG. 38 illustrates mutation detection on genomic or cfDNA using thebasic PCR-LDR-q PCR detection protocol with carryover prevention.Products are detected using TaqMan™ probes designed across the ligationjunction sequence.

FIG. 39 illustrates a variation of FIG. 38, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The PCR primers contain universal tails to eliminate primer dimerformation, and to allow for amplification with universal primers, one ofwhich contains a biotin, allowing for capture of products instreptavidin-coated wells. Ligation probes are hybridized to target andonly form product when there is perfect complementarity at the ligationjunction. Unreacted ligation probes, or target-independent ligationproducts are then washed away. LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection.

FIG. 40 illustrates a variation of FIG. 38, where the initialgene-specific PCR primers contain an RNA base, 4 additional bases and ablocking group on the 3′ end. These gene-specific primers are unblockedfrom the end by RNaseH2 only when hybridized to the target, liberating a3′OH end suitable for polymerase extension.

FIG. 41 illustrates a variation of FIG. 38, where the initialgene-specific PCR primers contain identical 8-11 base tails to preventprimer dimers. The upstream and downstream LDR probes contain UniAi andUniCi′ primer specific portions respectively. Further, the upstreammutation-specific LDR probes contain the mutation base at the 3′ end,followed by an RNA base and 4 more DNA bases that matches the target,and a C3 spacer to block ligation (or subsequent extension bypolymerase). Only in the presence of RNaseH2 and when hybridized to thecorrect target will the upstream blocking group be removed, liberating a3′OH end suitable for ligation. In this illustration, the upstream LDRprobe complementary to wild-type DNA also contains a blocking group, butno RNA base or 5′ tag. This probe further enhances ligation specificityin discriminating mutant from wild-type target.

FIG. 42 illustrates a variation of FIG. 41, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The PCR primers contain universal tails to eliminate primer dimerformation, and the LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection.

FIG. 43 illustrates a variation of FIG. 42, where the 5′ side of thedownstream LDR probe contains a base the same as the 3′ discriminatingbase on the upstream probe, said base removed by the 5′ to 3′ nucleaseactivity of Fen nuclease or Taq polymerase to liberate a 5′ phosphatesuitable for a subsequent ligation. The nuclease should only cleave whenthe downstream probe is hybridized to mutant target. Both the upstreamand downstream mutation-specific LDR probes contain short complementarysequences that only hybridize to each other when ligated together,generating FRET signal suitable for detection. In this illustration, theupstream LDR probe complementary to wild-type DNA does not contain acomplementary sequence, and would not generate a FRET signal even ifligated to a cleaved downstream probe.

FIG. 44 illustrates a variation of FIG. 43, where the initialgene-specific PCR primers contain identical 8-11 base tails to preventprimer dimers. The upstream and downstream LDR probes contain UniTaq Aiand UniTaq Bi′-UniTaq Ci′ primer specific and sequence tag portions.After ligation, the products are diluted and distributed into wellscontaining UniTaq-specific primers of the format UniTaq Ci and F1-UniTaqBi—Q—UniTaq Ai. (where F1 is a fluorescent dye that is quenched byQuencher Q). The product strand formed by the fluorescently labeledprimer will hairpin, such that the UniTaq Bi sequence pairs with theUniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye, and generatingsignal detected by a real-time PCR instrument.

FIG. 145 illustrates a variation of FIG. 41, where the PCR products areselectively amplified using mutation-selective upstream primers andlocus-specific downstream primers. Upon target-specific hybridization,RNaseH removes the mutant-specific RNA base to liberate a 3′OH groupsuitable for polymerase extension. RNaseH will preferentially cleave theRNA base when it is perfectly matched to mutant DNA, but will be lesslikely to cleave the RNA base when hybridized to wild-type DNA. Thisoccurs during every cycle of the PCR amplification, thus enriching foramplification of specific mutant targets. Optional primers withwild-type sequence lack the RNA base and remain blocked, thus furtherreducing amplification of wild-type sequence. After the initial PCRenrichment step, this procedure continues with LDR-q PCR detectionprotocol with carryover prevention. Products are detected using TaqMan™probes designed across the ligation junction sequence.

FIG. 146 illustrates a variation of FIG. 145, where the upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ primerspecific and sequence tag portions. After ligation, the products arediluted and distributed into wells containing UniTaq-specific primers ofthe format UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai, and signal generatedin the PCR is detected by a real-time PCR instrument.

FIG. 147 illustrates a variation of FIG. 145, where the PCR products arecaptured on a solid support. The LDR probes are designed to containshort complementary sequences that only hybridize to each other whenligated together, generating FRET signal suitable for detection.

FIG. 148 illustrates a variation of FIG. 41, where the PCR products areselectively amplified using locus-specific upstream and downstreamprimers. Upon target-specific hybridization, RNaseH removes the RNA baseto liberate a 3′OH group suitable for polymerase extension. A blockingLNA or PNA probe comprising wild-type sequence that partially overlapswith the upstream PCR primer will preferentially compete in binding towild-type sequence over the upstream primer, but not as much to mutantDNA, and thus suppresses amplification of wild-type DNA during eachround of PCR. After the initial PCR enrichment step, this procedurecontinues with LDR-qPCR detection protocol with carryover prevention.Products are detected using TaqMan™ probes designed across the ligationjunction sequence.

FIG. 149 illustrates a variation of FIG. 148, where the upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ primerspecific and sequence tag portions. After ligation, the products arediluted and distributed into wells containing UniTaq-specific primers ofthe format UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai, and signal generatedin the PCR is detected by a real-time PCR instrument.

FIG. 150 illustrates a variation of FIG. 148, where the PCR products arecaptured on a solid support. The LDR probes are designed to containshort complementary sequences that only hybridize to each other whenligated together, generating FRET signal suitable for detection.

FIG. 154 illustrates another variation where the PCR products areselectively amplified using locus-specific upstream primers that alsocomprise 5′ portion sequences complementary to wild-type sequence of thetop strand allowing for formation of loop-hairpins after extension, andlocus-specific downstream primers. Upon target-specific hybridization,RNaseH removes the RNA base to liberate a 3′OH group suitable forpolymerase extension. PCR is performed with a polymerase lacking 5′nuclease, 3′ nuclease, and strand-displacement activity. (i)Denaturation of wild-type bottom strand results in the formation of aloop-hairpin with perfect match at 3′ end, which is extended bypolymerase to form a longer hairpin region. This does not denature at72° C. and prevents upstream primer from generating a full-length topstrand. (ii) Denaturation of mutant bottom strand results in theformation of a loop-hairpin with mismatch at 3′ end. This is notextended by polymerase, and thus denatures at 72° C., enabling upstreamprimer to generate full-length top strand. (iii) Denaturation of topstrand results in hairpin on 5′ side, which denatures during the extendstep of PCR (72° C.), allowing polymerase to generate full-length bottomstrand. vThe difference in hairpin extension preference of upstreamprimers with (i) wild-type and (ii) mutant template results inpreferential amplification of mutant DNA. This selection againstamplification of wild-type DNA occurs during every cycle of the PCR,thus enriching for mutant targets. After the initial PCR enrichmentstep, this procedure continues with LDR-q PCR detection protocol withcarryover prevention. Products are detected using TaqMan™ probesdesigned across the ligation junction sequence.

FIG. 155 illustrates a variation of FIG. 154, where the PCR products arecaptured on a solid support. The LDR probes are designed to containshort complementary sequences that only hybridize to each other whenligated together, generating FRET signal suitable for detection.

As an example of quantitative mutation enumeration using spatialmultiplexing into 48 wells, consider total cfDNA in target sample thatis undergoing the initial PCR amplification is about 10,000 genomeequivalents, with 12 of those containing a mutation in KRAS codon 12.The initial distribution into 48 wells results in about 200 genomeequivalents per well, with 1 well containing 2 mutant copies, 10 wellscontaining 1 mutant copy, and the remaining 37 wells containing nomutant copies. After about 20 rounds of PCR amplification, (forsimplicity in calculation, say 99% efficiency of amplification, or about960,000-fold—complete efficiency would yield 1,046,576-foldamplification), then the total number of copies for the mutation will be960,000, and for the wild-type will be 192 million. Assuming a ligationefficiency of 50% on mutant DNA per cycle, times 20 cycles, and forligation on wild-type DNA, a ligation fidelity of 1,000-fold, then themutant DNA would yield 9.6 million molecules, while wild-type DNA wouldyield 1.9 million molecules. Spatial distribution into 48 wells wouldyield 200,000 and 40,000 molecules of LDR product for mutant andwild-type respectively. After addition of tag primers and TaqMan™ probewith real-time PCR, for simplicity in the calculations, the above LDRproducts convert to Ct values of 10 and 12.5 respectively. For anymutation-derived signal to be scored as positive, it would need toappear in at least 2 or 3 wells, and also easily distinguished from(low-level) signal arising from misligation of probes on wild-type DNA.Such mis-ligation to wild-type DNA may be even further suppressed byadding a wild-type upstream LDR probe, which would lack the fluorescentreporter, such that ligation products would be silent with no signal.The likely Poisson distribution (see e.g., FIG. 31) shows, the negativesample will have a range of distribution of (wells: molecules) (38:0;10:1; 1:2) for 12 molecules These numbers are sufficiently differentthat TaqMan™ readout will give quantitative enumeration of signal,allowing us to assign a score of 0, 1, or 2 original molecules per well(represented by Ct values of 12.5, 10, and 9 respectively), for a totalof 12 mutant KRAS molecules in the sample. In the case that the signalis only able to distinguish between 0 and 1 or more mutant molecules,given an initial 12 or fewer molecules enumerated in a minimum of 24wells, the initial number of mutant copies can be enumerated.

When using LDR-FRET detection, after distribution into the individual 48wells for solid-phase capture, assuming only 50% efficiency of capturingbiotinylated products, each well will have captured 10,000 mutant and 2million wild-type KRAS amplicons respectively. Assuming a ligationefficiency of only 50% on mutant template, at least 5,000 LDR productsshould be captured on the solid support if a single mutant molecule wasoriginally present. If two mutant molecules were in the original well,then approximately 10,000 LDR products should be captured. For the wellswith only wild-type product, assuming a ligation fidelity of 1:1,000(mutant upstream LDR probe misligated on wild-type DNA), only 1,000 LDRproducts would be captured. For any mutation-derived signal to be scoredas positive, it would need to appear in at least 2 or 3 wells, and alsoeasily distinguished from (low-level) signal arising from misligation ofprobes on wild-type DNA. Such mis-ligation to wild-type DNA may be evenfurther suppressed by adding a wild-type upstream LDR probe, which wouldlack the fluorescent reporter, such that ligation products would besilent with no signal. These numbers are sufficiently different thatLDR-FRET readout will give quantitative enumeration of signal, allowingassignment of a score of 0, 1, or 2 original molecules per well(represented as LDR-FRET signals of about 1,000, 5,000, and 10,000,respectively), for a total of 12 mutant KRAS molecules in the sample. Inthe case that the signal is only able to distinguish between 0 and 1 ormore mutant molecules, given an initial 12 or fewer molecules enumeratedin a minimum of 24 wells, the initial number of mutant copies can beenumerated.

When using UniTaq containing LDR probes, they are of the followingformat: Upstream probes comprise of a 5′ sequence tag, such as UniTaqAi,containing a primer specific portion, followed by target-specificsequence with a C:A or G:T mismatch at the 3^(rd) or penultimate base,the mutation base at the 3′ end, followed by an RNA base and 4 more DNAbases that matches the target, and a C3 spacer-blocking group. Thedownstream primers comprise a 5′ phosphorylated end, followed bytarget-specific sequence, and a 3′ sequence tag, such as UniTaqBi′—UniCi′, also containing a primer specific portion.

The LDR products may be detected using UniTaq-specific primers of theformat UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai (where F1 is a fluorescentdye that is quenched by Quencher Q). Under these conditions, thefollowing product will form: F1-UniTaq Bi—Q—UniTaq Ai—UpstreamTarget-Mutation-Downstream Target—UniTaq Bi′—UniTaq Ci′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye.

The initial PCR primers or upstream LDR probes may also contain an RNAbase, 4 additional bases and a blocking group (e.g. C3-spacer) on the 3′end. RNaseH2 is then added to the reaction. This assures that notemplate independent products are formed.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (i.e., 94°C. or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstream LDRprobe contains a base the same as the 3′ discriminating base on theupstream probe, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

Prophetic Example 2 High Sensitivity Methylation Marker for PromoterHypermethylation (When Present at 1% to 0.01%) in Total Plasma DNA.(e.g., p16 and Other Tumor Suppressor Genes, CpG “Islands” Also, Sept9,Vimentin, etc.)

Overview of approach v1: Isolated genomic DNA, or methyl enriched DNA istreated with a cocktail of methyl sensitive enzymes whose recognitionelements comprise only or mostly C and G bases (e.g.,Bsh1236I=CG{circumflex over ( )}CG; HinP1I=G{circumflex over ( )}CGC;AciI=CACGC or G{circumflex over ( )}CGG; and Hpy99I=CGWCGA). Judiciouslychosen PCR primers amplify uncut DNA fragments of about 100-130 bp. Thefragment should have at least 2-3 methyl sensitive enzyme sites, suchthat cleavage would cause these fragments to dissipate. These sites arechosen such that carryover prevention may work at two levels: (i) thesites are still cleavable in DNA containing incorporated dUTP, allowingfor use of UNG for carryover prevention and (ii) after amplification,the sites are unmethylated, such that products would readily berecleaved should they carryover to another reaction. Subsequent to theinitial PCR amplification, LDR and UniTaq reactions with carryoverprotection are performed as described above. Alternatively, LDR andTaqMan™, or straight TaqMan™ reactions may be performed to identify andquantify relative amounts of methylated DNA in the initial sample.

To summarize the levels of discrimination of the above approach usingboth PCR primers and LDR probes for detection of low-abundancemethylation:

-   1. Use of methylation sensitive restriction enzymes to cleave target    when not methylated.-   2. Use of PCR primers with universal tails, such that if any    target-independent primer dimer formed, the incorrect product will    form a hairpin that will inhibit further amplification.-   3. Use of UNG and methylation sensitive restriction enzymes to    prevent carryover contamination of initial PCR reaction.-   4. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   5. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   6. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed Protocol for Highly Sensitive Detection of Promoter    Methylation v1:

2.1.a. Incubate genomic DNA, cfDNA, or methyl enriched DNA in thepresence of Bsh1236I (CG{circumflex over ( )}CG) and HinP1I (GACGC), andUNG (37° C., 30-60 minutes) to completely digest unmethylated DNA andprevent carryover. Add buffer supplement to optimize multiplexed PCRamplification, dUTP, and other dNTP's, AmpliTaq Gold, and gene-specificprimers containing universal tails, such that if any target-independentprimer dimer formed, the incorrect product will form a hairpin that willinhibit further amplification. This initial genomic DNA—PCR reactionmixture is suitable for multiplex PCR amplification in 12, 24, 48, or 96individual wells (spatial multiplexing), or in a single well. Denaturedigested genomic DNA, inactivate UNG and restriction endonucleases, andactivate AmpliTaq Gold (94° C., 5-10 minute) and multiplex PCR amplifymutation containing fragments for a limited number of cycles (94° C., 10sec., 60° C. 30 sec., 72° C. 30 sec. for 16-20 cycles). The PCR primersare designed to have Tm values around 64-66° C., and will hybridizerobustly, even when used at concentrations 10 to 50-fold below the normfor uniplex PCR (10 nM to 50 nM each primer). The cycles are limited toretain relative balance of PCR products with respect to each other,while still amplifying low abundant sequences about 100,000 to1,000,000-fold. After PCR amplification, Taq polymerase is inactivated(by incubating at 99° C. for 30 minutes.)

2.1.b. Add thermostable ligase (preferably from strain AK16D), buffersupplement to optimized ligation conditions, and suitable upstream anddownstream LDR probes (10 nM to 20 nM each, downstream primers may besynthesized with 5′ phosphate, or kinased in bulk prior to reactions.Upstream probes comprise of a 5′ tag, such as UniAi followed bytarget-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniCi′. Perform 20 cycles of LDR, (94° C., 10 sec., 60° C. 4-5minutes). This will allow for ligation events to occur on the PCRproducts if methylated DNA was present in the original sample.

2.1.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR primers would be UniAi and UniCi respectively,and the products would be of the form:

UniAi—Upstream Target—Methylation Region—Downstream Target—UniCi′

Two or three fragments in a single promoter region may be interrogatedat the same time using the same fluorescent dye. The number ofmethylated fragments per promoter may be determined by total signal forthat dye. When using spatial multiplexing, the sample is distributed to12, 24, 48, or 96 individual wells prior to the 37° C. incubation step(but after addition of enzymes). In this manner, methylation across apromoter region of a given molecule of DNA may be distinguished frommethylation of three different regions on three different molecules.

Since there is no need to distinguish between a wild-type and a mutantsignal, the LDR step may be eliminated, with the initial PCR reactionfollowed directly by a secondary real-time PCR (e.g., TaqMan™) reaction.The disadvantage of going straight to a secondary PCR is that UNGcarryover protection would not be used since the initial PCR reactionproducts have incorporated dUTP, and thus would be destroyed by UNG. Oneapproach to address this problem would be to use standard dNTP's in theinitial PCR, and rely solely on the restriction endonucleases to destroyany potential carryover from the initial or subsequent PCR reactions,since these products are now unmethylated.

A second assay design is based on an initial restriction digestion, thenmultiplexed PCR amplification followed by distribution and capture ofPCR amplified targets on the wells of a microtiter plate. A single cycleof LDR enables capture of LDR products on the correct targets on thesolid support, while mis-ligations are washed away. The LDR products arequantified, either through LDR-FRET (qLDR), real-time PCR (qPCR), orother reporter systems.

FIG. 45 illustrates methylation detection on genomic or cfDNA using thebasic restriction digestion, PCR-LDR-q PCR detection protocol withcarryover prevention. Products are detected using TaqMan™ probesdesigned across the ligation junction sequence.

FIG. 46 illustrates a variation of FIG. 45, where the initialgene-specific PCR primers contain identical 8-11 base tails to preventprimer dimers. The upstream and downstream LDR probes contain UniTaq Aiand UniTaq Bi′-UniTaq Ci′ tags respectively. After ligation, theproducts are diluted and distributed into wells containingUniTaq-specific primers of the format UniTaq Ci and F1-UniTaqBi—Q—UniTaq Ai. (where F1 is a fluorescent dye that is quenched byQuencher Q). The product strand formed by the fluorescently labeledprimer will hairpin, such that the UniTaq Bi sequence pairs with theUniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye, and generatingsignal detected by a real-time PCR instrument.

FIG. 47 illustrates a variation of FIG. 45, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The PCR primers contain universal tails to eliminate primer dimerformation, and to allow for amplification with universal primers, one ofwhich contains a biotin, allowing for capture of products instreptavidin-coated wells. Ligation probes are hybridized to target andonly form product when there is perfect complementarity at the ligationjunction. Unreacted ligation probes, or target-independent ligationproducts are then washed away. LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection.

As an example of quantitative methylation enumeration using spatialmultiplexing into 48 wells, consider a sample of cfDNA with 12 genomeequivalents of tumor DNA, used to score for methylation on a marker onchromosome 20, which is amplified to about 4 copies per cancer cell.This would give us 48 copies of methylated DNA that is resistant todigestion. The unmethylated DNA is fragmented by the restriction enzyme,but a minority of unmethylated DNA survives, ˜12 copies, for a total of60 copies. Also, some age related methylation may occur, allow that torange to 12 copies. Accordingly, samples with no tumor-specificmethylation may have a signal in the range of 12-24 copies, while thosewith the marker on chromosome 20 methylated in all 4 chromosomes mayhave a total in the range of 60-72 copies. If one looks at the likelyPoisson distribution (see FIGS. 31 and 32, the negative sample will havea range of distribution of (wells: molecules) (38:0; 10:1; 1:2) for 12molecules to (29:0; 15:1; 4:2; 1:3) for 24 molecules, while the positivesamples will have a range of distribution of (14:0; 17:1; 11:2; 4:3;1:4) for 60 molecules to (11:0; 16:1; 12:2; 6:3; 2:4; 1:5) for 72molecules. The accuracy in distinguishing LDR signal arising from 2molecules (e.g., for LDR-TaqMan™, a Ct value of 9, or for LDR-FRETdetection 10,000 LDR products) from 3 molecules (e.g., for LDR-TaqMan™,a Ct value of 8.5, or for LDR-FRET 15,000 molecules) will depend on thestandard deviation of the LDR signal from well to well. Nevertheless,even if LDR signal is variable enough that distinguishing the higherlevel signals becomes too difficult, as long as the signal is cleanenough to distinguish 0, 1, and 2 initial molecules (represented asLDR-TaqMan™ Ct values of 12.5, 10, and 9, or LDR-FRET signals of about1,000, 5,000, and 10,000, respectively), this approach will have nodifficulty distinguishing and enumerating methylated signal arising fromthose individuals with authentic circulating tumor DNA, from those withage-related (but not cancerous) methylated signal in normal blood. Inthe case that the Ct or fluorescent signal is only able to distinguish 0from 1 or more initial methylated molecules, given an initial 12 orfewer genome equivalents of tumor DNA, and 60 or more genome equivalentsof methylated DNA for the particular region (e.g., chromsome 20)enumerated in a minimum of 48 wells, the approach should distinguish andenumerate methylated signal arising from those individuals withauthentic circulating tumor DNA from those with age-related (but notcancerous) methylated signal in normal blood.

When using UniTaq containing LDR probes, they are of the followingformat: Upstream probes comprise of a 5′ tag, such as UniTaqAi followedby target-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniTaq Bi′—UniTaq Ci′.

The LDR products may be detected using UniTaq-specific primers of theformat UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai. (where F1 is afluorescent dye that is quenched by Quencher Q). Under these conditions,the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Upstream Target—Methylation Region —DownstreamTarget—UniTaq Bi′—UniTaq Ci′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye.

The upstream LDR probes and PCR primers may also contain an RNA base, 4additional bases and a blocking group on the 3′ end. RNaseH2 is thenadded to the reaction. This assures that no template independentproducts are formed. In some designs, a methyl sensitive restrictionsite is downstream of the 3′ end of the PCR primer, such that cleavagewith the enzyme removes the binding sequence for the 4 additional bases,and cleavage of the RNA base by RNaseH2 is significantly reduced.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (e.g., 94°C. or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstreamprobe may contain a base the same as the 3′ discriminating base on theupstream probe, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

Overview of approach v2: As above, isolated genomic DNA, or methylenriched DNA is treated with a cocktail of methyl sensitive enzymes(HinP1I, Bsh1236I, AciI, Hpy99I, and HpyCH4IV), as well as by methylinsensitive enzymes (HaeIII and MspI). The idea is to generate afragment of DNA of approximately 40 bases or more, wherein the 5′phosphate of the fragment originated from a methyl insensitive enzyme.The fragment should have at least 2-3 methyl sensitive enzyme sites,such that cleavage would cause these fragments to dissipate. One strandof the genomic fragment is then hybridized onto an artificial templatecontaining a hairpin, with and upstream region, which is unrelated togenomic DNA, and can ligate to the genomic fragment at the 5′ phosphate.The single-stranded portion of the hairpin also contains a regioncomplementary to the target containing one or more methyl-sensitiverestriction enzyme sites. The same methyl sensitive enzymes are thenadded back in, and if an unmethylated target strand accidentally escapedthe initial restriction digestion step, it will be cleaved in thissecond step. A downstream oligonucleotide is added that hybridizes tothe genomic fragment, downstream of where it hybridized to the templatestrand. When extending the locus-specific primer, the 5→3′ exonucleaseactivity of polymerase destroys the template portion of the ligatedoligo, creating a product containing both upstream and downstream tagsand suitable for amplification. Unligated hairpin oligo will extend onitself and not amplify further. Both upstream and downstreamoligonucleotides have optional Universal sequences, as well as UniTaqspecific sequences, allowing for simultaneous “preamplification” for12-20 cycles, prior to opening tube, and dividing into the appropriateUniTaq or TaqMan™ assays. For each promoter region, there will be threepositions of interrogation, such that the signal appears (Ct valueindicating relative quantity of methylated sequence) as well as totalsignal strength (i.e. =1, 2, or 3 sites methylated for that promoter).This approach is also compatible with using UNG to provide carryoverprotection, and RNaseH2 to provide extra fidelity during the PCRamplification steps.

To summarize the levels of discrimination of the above approach fordetection of low-abundance methylation:

-   1. Use of methylation insensitive restriction enzyme to generate a    unique 5′ phosphate on double-stranded target DNA.-   2. Use of methylation sensitive restriction enzymes to cleave    double-stranded target when not methylated.-   3. Use of UNG and methylation sensitive restriction enzymes to    prevent carryover contamination of initial PCR reaction.-   4. Use of ligation fidelity of thermostable ligase to ligate correct    tag to target strand.-   5. Use of locus specific primer and polymerase to amplify ligated    target strands.-   6. Use of nuclease activity of RNaseH2 to liberate an unblocked 3′    OH on the PCR primers, only when hybridized to target.-   7. Use of sequences on the 3′ end of tag oligos, such that when they    are not ligated, form hairpins and extend on themselves to form    products that do not amplify.-   8. Use of UniTaq or tag primers to amplify PCR or LDR products for    real-time PCR readout.    Detailed Protocol for Highly Sensitive Detection of Promoter    Methylation v2:

2.2a. Prepare mix containing restriction enzymes, artificial hairpintemplates (see below), and thermostable ligase. Cleave isolated genomicDNA, or methyl enriched DNA with a cocktail of methyl sensitive enzymes(e.g. HinP1I, Bsh1236I, AciI, Hpy99I, and HpyCH4IV), as well as bymethyl insensitive enzymes (HaeIII and MspI). Generate fragments ofapproximately 40 bases or more that have a 5′ phosphate from a HaeIII orMspI site, and at least 3 methyl sensitive sites (that are not cleavedbecause they were methylated). Preferably, generate three such fragmentsper promoter. Heat-kill endonucleases (65° C. for 15 minutes) anddenature DNA (94° C. 1 minute). Artificial templates contain upstreamprimer region (optional 5′ Universal Primer U1Pm, followed by UniTaq Ai)as well as a region complementary to UniTaq Ai, and a regioncomplementary to target DNA with Tm of about 72° C., and overlap with atleast one methyl-sensitive restriction site). Incubate at 60° C. toallow for hybridization and ligation of hairpin oligonucleotides to 5′phosphate of target DNA if and only if it was methylated and hybridizedto the correct template. This initial genomic DNA—ligation productmixture is suitable for multiplex PCR amplification in 12, 24, 48, or 96individual wells (spatial multiplexing), or in a single well.

2.2b. Add: The methyl-sensitive restriction enzymes (incubate at 37° C.for 30 minutes), as well as Hot-start Taq polymerase, dNTPs, UniTaqAi,and downstream primers (containing 5′ Universal Primer U2, followed byUniTaq Bi, followed by target locus-specific sequence complementary tothe target fragment with sequence that is just downstream of theartificial template strand sequence). When extending the locus-specificprimer, the 5′→3′ exonuclease activity of polymerase destroys thetemplate portion of the ligated oligonucleotide, creating a productcontaining both upstream and downstream tags and suitable foramplification. Unligated hairpin oligo will extend on itself and notamplify further. Ideally, the universal primer tails U1Pm and U2 on theLDR and PCR compound primers are slightly shorter than Universal primersU1 and U2. This allows initial universal amplification at a lowercycling temperature (e.g., 55° C. annealing) followed by higher cyclingtemperature (e.g., 65° C. annealing) such that the universal primersU1Pm and U2 bind preferentially to the desired product (compared tocomposite primers binding to incorrect products). In the preferredvariation to minimize target independent amplifications, the downstreamPCR primers contain an RNA base and a blocked 3′ end, which is liberatedby an RNase-H that cleaves the RNA base when the primer is hybridized toits target. These conditions amplify products of the sequence:

Univ. Primer U1Pm—UniTaq Ai —Methylation Region—UniTaq Bi′—Univ. PrimerU2′

Or simply of the sequence:

UniTaq Ai—Methylation Region—UniTaq Ci′

2.2c. Open tube, dilute 10- to 100-fold and distribute aliquots toTaqMan™ wells, each well containing the following primers: UniversalPrimer U2 and UniTaq specific primers of the format F1-UniTaqBi—Q—UniTaq Ai. (where F1 is a fluorescent dye that is quenched byQuencher Q). Under these conditions, the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Methylation Region—UniTaq Bi′—Univ.Primer U2′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When Universal Primer U2 binds to theUniv.Primer U2′ sequence, the 5′→3′ exonuclease activity of polymerasedigests the UniTaq Bi sequence, liberating the F1 fluorescent dye.

For products of the sequence UniTaq Ai—Target DNA—UniTaq Ci′, they maybe detected using nested PCR primers, with the optional RNaseH2 cleavageto remove blocking groups, and an internal TaqMan™ probe.

As a control for the total amount of DNA present, one can choose anearby target fragment where the 5′ phosphate is generated by a methylinsensitive enzyme (HaeIII or MspI), and the rest of the fragment islacking in methyl sensitive enzyme sites. The upstream oligonucleotidethat is ligated to the target fragment is a mixture of two oligos: (i)An oligonucleotide present at 1 in 100 with the correct UniTaq specificsequence, and (ii) an oligonucleotide present at 99 in 100 with asequence that has about 8-10 bases complementary to its 3′ end. Afterthe ligation event and destroying template with UNG and AP endonuclease,the universal primers are added for PCR amplification. The ligationproduct containing the UniTaq sequences amplifies and will give a signalequivalent to 1 in 100 of the original template. The majority ligationproduct lacks the universal sequence on the 5′ end, and does not amplifyexponentially. Unligated upstream probe will form a hairpin back onitself, and extend its own 3′ sequence on itself, taking it out ofcontention for becoming part of another PCR amplicon. Alternatively orin addition, the control may use a different ratio of the twooligonucleotides, for example 1:10 or 1:1,000 to allow for accuratecomparisons to low-levels of the methylated DNA present at the promotersite of interest.

An alternative control uses a mixture of two oligos: (i) A hairpinoligonucleotide present at 1 in 100 with the correct UniTaq specificsequence, and (ii) A hairpin oligonucleotide present at 99 in 100without the UniTaq sequence. After the ligation event, the universalprimers are added for PCR amplification. When extending thelocus-specific primer, the 5′→3′ exonuclease activity of polymerasedestroys the template portion of the ligated oligo, creating a productcontaining both upstream and downstream tags and suitable foramplification. Unligated hairpin oligo will extend on itself and notamplify further. The ligation product containing the UniTaq sequencesamplifies and will give a signal equivalent to 1 in 100 of the originaltemplate. The majority ligation product lacks the universal sequence onthe 5′ end, and does not amplify exponentially.

FIG. 48 illustrates methylation detection on genomic or cfDNA using therestriction digestion, hairpin ligation, locus-specific extension, withPCR amplification protocol with carryover prevention. Products aredetected using nested PCR primers with TaqMan™ probes designed acrossthe methylated sequence.

FIG. 49 illustrates a variation of FIG. 48, where the initialgene-specific PCR primers contain tags UniAi and UniCi. The products arediluted and distributed into wells for a nested amplification. Theupstream and downstream nested PCR primers contain UniTaq Aj and UniTaqBj-UniTaq Cj tags on their 5′ ends, respectively. In addition,UniTaq-specific primers of the format UniTaq Cj and F1-UniTaqBj—Q≥UniTaq Aj are present in the amplification mix at higherconcentration, and thus become the dominant primers incorporating intothe amplification products. The product strand formed by thefluorescently labeled primer will hairpin, such that the UniTaq Bjsequence pairs with the UniTaq Bj′ sequence. When UniTaq Primer Cj bindsto the UniTaq Cj′ sequence, the 5′→3′ exonuclease activity of polymerasedigests the UniTaq Bj sequence, liberating the F1 fluorescent dye, andgenerating signal detected by a real-time PCR instrument.

The products of the sequence UniTaq Ai—Target DNA—UniTaq Ci′ may also bedetected using primers nested inside the UniTaq Ci and UniTaq Aisequence, and a standard TaqMan™ probe.

Two or three fragments in a single promoter region may be interrogatedat the same time using the same fluorescent dye. The number ofmethylated fragments per promoter may be determined by total signal forthat dye. When using spatial multiplexing, the sample is distributed to12, 24, 48, or 96 individual wells prior to the 37° C. incubation step(but after addition of enzymes). In this manner, methylation across apromoter region of a given molecule of DNA may be distinguished frommethylation of three different regions on three different molecules.

Since there is an initial ligation step, a subsequent LDR step is notnecessary, with the initial PCR reaction followed directly by asecondary real-time PCR (e.g., TaqMan™) reaction. The disadvantage ofgoing straight to a secondary PCR is that UNG carryover protection wouldnot be used since the initial PCR reaction products have incorporateddUTP, and thus would be destroyed by UNG. One approach to address thisproblem would be to use standard dNTP's in the initial PCR, and relysolely on the restriction endonucleases to destroy any potentialcarryover from the initial or subsequent PCR reactions, since theseproducts are now unmethylated.

The PCR primers may also contain an RNA base, 4 additional bases and ablocking group on the 3′ end. RNaseH2 is then added to the reaction.This assures that no template independent products are formed with theUniTaq primer sets. In some designs, a methyl sensitive restriction siteis downstream of the 3′ end of the PCR primer, such that cleavage withthe enzyme removes the binding sequence for the 4 additional bases, andcleavage of the RNA base by RNaseH2 is significantly reduced.

Overview of approach v3: Isolated genomic DNA, or methyl enriched DNA istreated with a methyl sensitive enzymes whose recognition elementscomprise only of CpG dinucleotide pairs (i.e. Bsh1236I=CG{circumflexover ( )}CG; and Hpy99I=CGWCGA). Treat with bisulfite, which converts“dC” to “dU”, and renders the strands non-complementary. Hybridizelocus-specific primers in the presence of BstU1 (CGACG), which willcleave carryover DNA. Primers and target that were not cleaved areunblocked with RNaseH2 only when bound to target. Unblocked PCR primersthen amplify uncut bisulfite-converted DNA fragments of about 100-130bp. The fragment should have at least 2 methyl sensitive enzyme sites,such that cleavage would cause these fragments to dissipate. These sitesare chosen such that carryover prevention may work at two levels: (i)the sites are still cleavable in DNA containing incorporated dUTP,allowing for use of UNG for carryover prevention and (ii) afteramplification, the sites are unmethylated, such that products wouldreadily be recleaved should they carryover to another reaction. Further,the fragment should have additional internal methylated CpG pairs, suchthat a blocking primer would enrich for amplification of initiallymethylated target, and further the LDR probes would also select fordetection of initially methylated target. Subsequent to the initial PCRamplification, LDR and UniTaq reactions with carryover protection areperformed as described above. Alternatively, LDR and TaqMan™, orstraight TaqMan™ reactions may be performed to identify and quantifyrelative amounts of methylated DNA in the initial sample.

To summarize the levels of discrimination of the above approach usingboth PCR and LDR primers for detection of low-abundance methylation:

-   1. Use of methylation sensitive restriction enzymes to cleave target    when not methylated.-   2. Use of nuclease activity of RNaseH2 to liberate an unblocked 3′    OH on the PCR primers, only when hybridized to target.-   3. Use of UNG and methylation sensitive restriction enzymes to    prevent carryover contamination of initial PCR reaction.-   4. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   5. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   6. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed Protocol for Highly Sensitive Detection of Promoter    Methylation v3

2.3.a. Incubate genomic DNA, or methyl enriched DNA in the presence ofBsh1236I (CGACG) and UNG (37° C., 30-60 minutes) to completely digestunmethylated DNA and prevent carryover. Treat with bisulfite, whichrenders the strands non-complementary, and purify DNA strands using acommercially available kit (i.e. from Zymo Research or Qiagen). Addbuffer supplement to optimize multiplexed PCR amplification, dUTP, andother dNTP's, AmpliTaq Gold, RNaseH2, BstU1 (CG{circumflex over ( )}CG),and gene-specific primers containing an RNA base after the desired 3′end, 4 additional bases, and a blocking group to prevent extension onincorrect targets. This initial genomic DNA—PCR reaction mixture issuitable for multiplex PCR amplification in 12, 24, 48, or 96 individualwells (spatial multiplexing), or in a single well. BstU1 will cleave anycarryover DNA from an earlier amplification. Denature digested genomicDNA and the restriction endonuclease, and activate AmpliTaq Gold (94°C., 5-10 minute) and multiplex PCR amplify mutation containing fragmentsfor a limited number of cycles (94° C., 10 sec., 60° C. 30 sec., 72° C.30 sec. for 16-20 cycles). The PCR primers are designed to have Tmvalues around 60° C., but with the 5 extra bases they are closer to65-68° C., and will hybridize robustly, even when used at concentrations10 to 50-fold below the norm for uniplex PCR (10 nM to 50 nM eachprimer). In addition, a blocking oligonucleotide is used to limitamplification of unmethylated DNA. The cycles are limited to retainrelative balance of PCR products with respect to each other, while stillamplifying low abundant sequences about 100,000 to 1,000,000—fold. AfterPCR amplification, Taq polymerase is inactivated (by incubating at 99°C. for 30 minutes.)

2.3.b. Add thermostable ligase (preferably from strain AK16D), buffersupplement to optimized ligation conditions, and suitable upstream anddownstream LDR probes (10 nM to 20 nM each, downstream probes may besynthesized with 5′ phosphate, or phosphorylated with kinase in bulkprior to reactions. Upstream probes comprise of a 5′ tag, such as UniAifollowed by target-specific sequence. The downstream probes comprise a5′ phosphorylated end, followed by target-specific sequence, and a 3′tag, such as UniCi′. Perform 20 cycles of LDR, (94° C., 10 sec., 60° C.4-5 minutes). This will allow for ligation events to occur on the PCRproducts if methylated DNA was present in the original sample.

2.3.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR probes would be UniAi and UniCi respectively,and the products would be of the form:

UniAi—Upstream Target—Bisulfite-converted Methylation—egion —DownstreamTarget—UniCi′

Two or three fragments in a single promoter region may be interrogatedat the same time using the same fluorescent dye. The number ofmethylated fragments per promoter may be determined by total signal forthat dye. When using spatial multiplexing, the sample is distributed to12, 24, 48, or 96 individual wells prior to the 37° C. incubation step(but after addition of enzymes). In this manner, methylation across apromoter region of a given molecule of DNA may be distinguished frommethylation of three different regions on three different molecules.

FIG. 50 illustrates methylation detection on genomic or cfDNA usingrestriction digestion, and bisulfate treatment. The initialbisulfite-converted methylated region PCR primers contain an RNA base, 4additional bases and a blocking group on the 3′ end. Hybridization ofthese primers in the presence of BstU1 will cleave carryover DNA. Thesegene-specific primers are unblocked from the end by RNaseH2 only whenhybridized to the target, liberating a 3′OH end suitable for polymeraseextension. A blocking oligonucleotide is used to limit amplification ofbisulfate-converted, unmethylated DNA. LDR probes forbisulfate-converted methylated DNA target provide additionalspecificity. Products are detected using TaqMan™ probes designed acrossthe ligation junction sequence.

FIG. 51 illustrates a variation of FIG. 50. The upstream and downstreamLDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively. After ligation, the products are diluted and distributedinto wells containing UniTaq-specific primers of the format UniTaq Ciand F1-UniTaq Bi—Q—UniTaq Ai (where F1 is a fluorescent dye that isquenched by Quencher Q). The product strand formed by the fluorescentlylabeled primer will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye, and generatingsignal detected by a real-time PCR instrument.

FIG. 52 illustrates a variation of FIG. 50, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The PCR primers contain universal tails to eliminate primer dimerformation, and to allow for amplification with universal primers, one ofwhich contains a biotin, allowing for capture of products instreptavidin-coated wells. Ligation probes are hybridized to target andonly form product when there is perfect complementarity at the ligationjunction. Unreacted ligation probes, or target-independent ligationproducts are then washed away. LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection.

FIG. 151 illustrates a variation of FIG. 50, where after initialrestriction endonuclease digestion and bisulfite conversion, the PCRproducts are selectively amplified using locus-specific upstream anddownstream primers. Upon target-specific hybridization, RNaseH removesthe RNA base to liberate a 3′OH group suitable for polymerase extension.A blocking LNA or PNA probe comprising bisulfite converted unmethylatedsequence (or its complement) that partially overlaps with the upstreamPCR primer will preferentially compete in binding to bisulfite convertedunmethylated target sequence over the upstream primer, but not as muchto bisulfite converted methylated target sequence, and thus suppressesamplification of bisulfite converted unmethylated target sequence duringeach round of PCR. After the initial PCR enrichment step, this procedurecontinues with LDR-qPCR detection protocol with carryover prevention.Products are detected using TaqMan™ probes designed across the ligationjunction sequence.

FIG. 152 illustrates a variation of FIG. 151, where the upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ primerspecific and sequence tag portions. After ligation, the products arediluted and distributed into wells containing UniTaq-specific primers ofthe format UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai, and signal generatedin the PCR is detected by a real-time PCR instrument.

FIG. 153 illustrates a variation of FIG. 151, where the PCR products arecaptured on a solid support. The LDR probes are designed to containshort complementary sequences that only hybridize to each other whenligated together, generating FRET signal suitable for detection.

FIG. 156 illustrates a variation of FIG. 50, where the PCR products areselectively amplified using locus-specific upstream primers that alsocomprise 5′ portion sequences complementary to bisulfite-treatedunmethylated sequence of the top strand allowing for formation ofloop-hairpins after extension, and locus-specific downstream primers.Upon target-specific hybridization, RNaseH removes the RNA base toliberate a 3′OH group suitable for polymerase extension. PCR isperformed with a polymerase lacking 5′ nuclease, 3′ nuclease, andstrand-displacement activity. (i) Denaturation of bisulfite-treatedunmethylated bottom strand results in loop-hairpin with perfect match at3′ end, which is extended by polymerase to form a longer hairpin region.This does not denature at 72° C. and prevents upstream primer fromgenerating full-length top strand. (ii) Denaturation ofbisulfite-treated methylated bottom strand results in loop-hairpin withtwo or more mismatches. This is not extended by polymerase, and thusdenatures at 72° C., enabling upstream primer to generate full-lengthtop strand. (iii) Denaturation of top strand results in hairpin on 5′side, which denatures during the extend step of PCR (72° C.), allowingpolymerase to generate full-length bottom strand. The difference inhairpin extension preference of upstream primers with (i)bisulfite-treated unmethylated template and (ii) bisulfite-treatedmethylated template results in preferential amplification of mutant DNA.This selection against amplification of bisulfate-treated unmethylatedtarget occurs during every cycle of the PCR, thus enriching forbisulfate-treated methylated targets. After the initial PCR enrichmentstep, this procedure continues with LDR-q PCR detection protocol withcarryover prevention. Products are detected using TaqMan™ probesdesigned across the ligation junction sequence.

Figure illustrates a variation of FIG. 148, where the PCR products arecaptured on a solid support. The LDR probes are designed to containshort complementary sequences that only hybridize to each other whenligated together, generating FRET signal suitable for detection.

The upstream LDR probes and PCR primers may also contain an RNA base, 4additional bases and a blocking group on the 3′ end. RNaseH2 is thenadded to the reaction. This assures that no template independentproducts are formed. In some designs, a methyl sensitive restrictionsite is downstream of the 3′ end of the PCR primer, such that cleavagewith the enzyme removes the blocking group.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (e.g., 94°C. or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstreamprimer may contain a base the same as the 3′ discriminating base on theupstream primer, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

FIG. 53 illustrates a variation of FIG. 50, where the PCR products aredistributed (spatial multiplexing), and then subjected to a secondnested PCR using a TaqMan™ probe as well as a blocking oligonucleotideto limit amplification of bisulfite-converted, unmethylated DNA.

FIG. 151 158 illustrates a variation of FIG. 45, where the primary PCRproducts are detected directly using nested locus-specific primers andinternal TaqMan™ probes. PCR products incorporate dU, and areunmethylated, allowing for carryover prevention.

Prophetic Example 3 High Sensitivity Detection of Gene Translocation orSplice-Site Variation in mRNA Isolated from Total Plasma mRNA, Exosomes,Circulating Tumor Cells (CTC's) or Total Blood Cells Containing CTC's

Overview of approach: This approach depends on the fidelity of threeenzymes: (i) reverse transcriptase and faithfully copy low-level copiesof aberrant RNA transcripts in the initial sample, (ii) Taq polymeraseto proportionally amplify the cDNA, and (iii) thermostable ligase indiscriminating primers hybridized adjacent to each other. Once aligation event has taken place, those products will be amplified in asubsequent Real-time PCR amplification step, and thus this is the keydiscriminatory step.

One advantage of using LDR is that it can discriminate a translocationevent independent of the precise breakpoints. Further, when atranslocation or alternative splicing creates new exon-exon junctions,LDR is ideally suited to precisely distinguish these junctions, down tothe exact bases at the junctions.

There are at least two sources of aberrantly spliced transcripts intumors. Tumors may undergo global dysregulation of gene expressionthrough overall hypo-methylation. One consequence of hypomethylation isthe degradation of control of transcription start sites in promoterregions, allowing for alternative sequences in the 5′ end oftranscripts. Such alternatively spliced leader sequences may then beaccurately identified and quantified using LDR-based assays. A secondsource of aberrantly spliced transcripts arises from dysregulation ofthe splicing machinery. Some such transcripts are translated intoproteins that facilitate or even drive tumor growth. Again, thesealternatively spliced transcripts may then be accurately identified andquantified using LDR-based assays, including providing relative levelsof both the aberrant and wild-type transcript in the same LDR reaction.

To protect against carryover contamination, UNG is added to the reactionprior to polymerase activation, and the initial PCR amplification isperformed with dUTP. The LDR probes are comprised of the natural bases,thus the LDR product is now resistant to UNG digestion in the secondreal-time PCR step. Note that the LDR products contain tags or UniTaqsequences on their non-ligating ends, which are lacking in the targetDNA, thus accidental carryover of LDR products does not result inlarge-scale amplification. Unlike with PCR, an initial LDR product isnot a substrate for a second LDR reaction.

To summarize the levels of discrimination of the above approach for highsensitivity detection of translocation or splice-site variation in mRNA:

-   1. Use of PCR primers with universal tails, such that if any    target-independent primer dimer formed, the incorrect product will    form a hairpin that will inhibit further amplification.-   2. Use of UNG to prevent carryover contamination of initial PCR    reaction.-   3. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   4. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   5. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed Protocol for Highly Sensitive Detection of Gene    Translocation or Splice-Site Variation in mRNA

3.1.a. Incubate isolated mRNA (or even total isolated nucleic acids) inthe presence of UNG (37° C., 15-30 minutes, to prevent carryover), dUTP,and other dNTP's, MMLV reverse transcriptase, AmpliTaq Gold, andtranscript-specific primers. (MMLV reverse transcriptase may beengineered to synthesize cDNA at 50-60° C., from total input RNA(Invitrogen Superscript III). Alternatively, Tth or Tma DNA polymeraseshave been engineered to improve their reverse-transcriptase activity(may require addition of Mn cofactor). Finally, thermophilic PyroPhage3173 DNA Polymerase has both strand-displacement andreverse-transcription activity, and may also be used.) This initialcDNA—PCR reaction mixture is suitable for multiplexreverse-transcription PCR amplification in 12, 24, 48, or 96 individualwells (spatial multiplexing), or in a single well. After extension ofreverse primers on their cognate RNA transcripts to generate cDNA,inactivate UNG and MMLV reverse transcriptase, and activate AmpliTaqGold (94° C., 5-10 minute) and multiplex PCR amplifytranscript-containing fragments for a limited number of cycles (94° C.,10 sec., 60° C. 30 sec., 72° C. 30 sec. for 16-20 cycles). The PCRprimers are designed to have Tm values around 64-66° C., and willhybridize robustly, even when used at concentrations 10 to 50-fold belowthe norm for uniplex PCR (10 nM to 50 nM each primer). The cycles arelimited to retain relative balance of PCR products with respect to eachother, while still amplifying low abundant sequences about 100,000 to1,000,000—fold. After PCR amplification, Taq polymerase is inactivated(by incubating at 99° C. for 30 minutes.)

3.1.b. Add thermostable ligase (preferably from strain AK16D), buffersupplement to optimized ligation conditions, and suitable upstream anddownstream LDR probes (10 nM to 20 nM each, downstream probes may besynthesized with 5′ phosphate, or phosphorylated with kinase in bulkprior to reactions; upstream probes comprise of a 5′ tag, such as UniAifollowed by transcript-specific sequence. The downstream probes comprisea 5′ phosphorylated end, followed by target-specific sequence, and a 3′tag, such as UniCi′. Perform 20 cycles of LDR, (94° C., 10 sec., 60° C.4-5 minutes). This will allow for ligation events to occur on the cDNAPCR products if the desired exon-exon junction is present. For detectionof translocations where the precise junction is unknown, three LDRprobes are used. The middle probe (s) contains sequence complementary tothe known upstream and downstream regions of the (various) splicedtranscripts. The upstream and downstream probes contain tags asdescribed above to enable subsequent UniTaq or TaqMan™ amplification anddetection of the desired ligation products.

3.1.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR probes would be UniAi and UniCi respectively,and the products would be of the form:

UniTaq Ai—Upstream Exon—Downstream Exon Junction—UniTaq Ci′

For products using three LDR probes to detect transcripts with unknownjunctions, the following product will form:

UniTaq Ai—Upstream Exon—Bridge sequence—Downstream Exon—UniTaq Ci′

FIG. 54 illustrates an overview of the approach to use PCR-LDR reactionwith carryover prevention to detect translocation at the mRNA level. Anillustration of a translocation between two genes is shown, with thecrossover allowing exons 1, 2, or 3 of the upstream gene to fuse withexon b of the downstream gene. LDR probes are designed to detect all thepossible exon junctions (1-b, 2-b, and 3-b).

FIG. 55 illustrates a close up of translocation detection (overview inFIG. 54) on a fusion gene using the basic RT-PCR-LDR-qPCR detectionprotocol with carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers. Products are detected using TaqMan™ probesdesigned across the ligation junction sequence.

FIG. 56 illustrates a variation of FIG. 55, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primers.

FIG. 57 illustrates a variation of FIG. 55, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal suitable for detection.

FIG. 58 illustrates an overview of the approach to use PCR-LDR reactionwith carryover prevention to detect alternative splicing. Anillustration of an example of normal (1-2-3a-4) and alternative splicevariant (1-2-3b-4) mRNA's are illustrated. LDR probes are designed todetect both the normal and/or the alternative splice variant (3a-4, and3b-4).

FIG. 59 illustrates a close up of alternative splice variant detection(overview in FIG. 58) using the basic RT-PCR-LDR-qPCR detection protocolwith carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers. Products are detected using differentlylabeled TaqMan™ probes designed across the ligation junction sequences,for the normal transcript 3a-4 (F1), and for the alternative splicevariant 3b-4 (F2).

FIG. 60 illustrates a variation of FIG. 59, where upstream anddownstream LDR probes contain UniTaq Ai or UniTaq Aj and UniTaqBi′-UniTaq Ci′ tags respectively, and products are detected usingfluorescently labeled UniTaq primers F1-UniTaq Bi—Q—UniTaq Ai andF2-UniTaq Bi—Q—UniTaq Aj to detect signals F1 and F2, representingnormal transcript 3a-4, and for the alternative splice variant 3b-4,respectively.

FIG. 61 illustrates a variation of FIG. 59, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 and F2 suitable for detection, representing normal transcript3a-4, and for the alternative splice variant 3b-4, respectively.

FIG. 62 illustrates a close up of low-level alternative splice variantdetection (overview in FIG. 58) using the basic RT-PCR-LDR-q PCRdetection protocol with carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers, and are designed to amplify only the minoritytranscript containing the 3b-4 junction. Products are detected usingTaqMan™ probes designed across the ligation junction sequence for thelow-abundant alternative splice variant 3b-4.

FIG. 63 illustrates a variation of FIG. 62, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal F1, representinglow-abundant alternative splice variant 3b-4.

FIG. 64 illustrates a variation of FIG. 62, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 suitable for detection, representing the low-abundantalternative splice variant 3b-4.

FIG. 65 illustrates an overview of the approach to use PCR-LDR reactionwith carryover prevention to detect alternative splicing, with analternative start site for the first exon. An illustration of an exampleof normal (1-2-3) and alternative splice variant (1a-2-3) mRNAs areshown. LDR probes are designed to detect both the normal and/or thealternative splice variant (1-2, and 1a-2).

FIG. 66 illustrates a close up of alternative splice variant detection(overview in FIG. 65) using the basic RT-PCR-LDR-qPCR detection protocolwith carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers. Products are detected using differentlylabeled TaqMan™ probes designed across the ligation junction sequences,for the normal transcript 1-2 (F1), and for the alternative start sitevariant 1a-2 (F2).

FIG. 67 illustrates a variation of FIG. 66, where upstream anddownstream LDR probes contain UniTaq Ai or UniTaq Aj and UniTaqBi′-UniTaq Ci′ tags respectively, and products are detected usingfluorescently labeled UniTaq primers F1-UniTaq Bi—Q—UniTaq Ai andF2-UniTaq Bi—Q—UniTaq Aj to detect signals F1 and F2, representingnormal transcript 1-2, and for the alternative start site variant 1a-2,respectively.

FIG. 68 illustrates a variation of FIG. 66, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 and F2 suitable for detection, representing normal transcript1-2, and for the alternative start site variant 1a-2, respectively.

FIG. 69 illustrates a close up of low-level alternative splice variantdetection (overview in FIG. 58) using the basic RT-PCR-LDR-q PCRdetection protocol with carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers, and are designed to amplify only the minoritytranscript containing the 1a-2 junction. Products are detected usingTaqMan™ probes designed across the ligation junction sequence for thelow-abundant alternative start site variant 1a-2.

FIG. 70 illustrates a variation of FIG. 69, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal F1, representinglow-abundant alternative start site variant 1a-2.

FIG. 71 illustrates a variation of FIG. 69, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 suitable for detection, representing the low-abundantalternative start site variant 1a-2.

FIG. 72 illustrates an overview of the approach to use PCR-LDR reactionwith carryover prevention to detect alternative splicing with exondeletion. An illustration of an example of normal (e1-e2-e3-e4-e5) andalternative splice variant (e1-e2-e3-e5) mRNA's are illustrated. LDRprobes are designed to detect both the normal and/or the alternativesplice exon-deletion variant (e4-e5, and e3-e5).

FIG. 73 illustrates a close up of alternative splice variant (exondeletion) detection (overview in FIG. 58) using the basic RT-PCR-LDR-qPCR detection protocol with carryover prevention. The initialgene-specific reverse-transcription and PCR primers contain identical8-11 base tails to prevent primer dimers. Products are detected usingdifferently labeled TaqMan™ probes designed across the ligation junctionsequences, for the normal transcript e4-e5 (F1), and for the alternativesplice exon-deletion variant e3-e5 (F2).

FIG. 74 illustrates a variation of FIG. 73, where upstream anddownstream LDR probes contain UniTaq Ai or UniTaq Aj and UniTaqBi′-UniTaq Ci′ tags respectively, and products are detected usingfluorescently labeled UniTaq primers F1-UniTaq Bi—Q—UniTaq Ai andF2-UniTaq Bi—Q—UniTaq Aj to detect signals F1 and F2, representingnormal transcript e4-e5, and for the alternative splice exon-deletionvariant e3-e5, respectively.

FIG. 75 illustrates a variation of FIG. 73, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 and F2 suitable for detection, representing normal transcripte4-e5, and for the alternative splice exon-deletion variant e3-e5,respectively.

FIG. 76 illustrates a close up of low-level alternative splice variant(exon deletion) detection (overview in FIG. 58) using the basicRT-PCR-LDR-q PCR detection protocol with carryover prevention. Theinitial gene-specific reverse-transcription and PCR primers containidentical 8-11 base tails to prevent primer dimers, and are designed toamplify only the minority transcript containing the e3-e4 junction byusing a blocking oligonucleotide with e4 sequence to suppressamplification of wild-type transcript. Products are detected usingTaqMan™ probes designed across the ligation junction sequence for thelow-abundant alternative splice exon-deletion variant e3-e5.

FIG. 77 illustrates a variation of FIG. 76, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal F1, representinglow-abundant alternative splice exon-deletion variant e3-e5.

FIG. 78 illustrates a variation of FIG. 76, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 suitable for detection, representing the low-abundantalternative splice exon-deletion variant e3-e5.

FIG. 79 illustrates an overview of the approach to use PCR-LDR reactionwith carryover prevention to detect alternative splicing with introninsertion. An illustration of an example of normal (e1-e2-e3-e4-e5) andalternative splice variant (e1-i1-e2-e3-e4-e5) mRNA's are shown. LDRprobes are designed to detect both the normal and/or the alternativesplice variant with intron insertion (e1-e2, and i1-e2).

FIG. 80 illustrates a close up of alternative splice variant detection(overview in FIG. 79) using the basic RT-PCR-LDR-real-time PCR detectionprotocol with carryover prevention. The initial gene-specificreverse-transcription and PCR primers contain identical 8-11 base tailsto prevent primer dimers. Products are detected using differentlylabeled TaqMan™ probes designed across the ligation junction sequences,for the normal transcript e1-e2 (F1), and for the alternative splicevariant with intron insertion i1-e2 (F2).

FIG. 81 illustrates a variation of FIG. 80, where upstream anddownstream LDR probes contain UniTaq Ai or UniTaq Aj and UniTaqBi′-UniTaq Ci′ tags respectively, and products are detected usingfluorescently labeled UniTaq primers F1-UniTaq Bi—Q—UniTaq Ai andF2-UniTaq Bi—Q—UniTaq Aj to detect signals F1 and F2, representingnormal transcript e1-e2, and for the alternative splice variant withintron insertion i1-e2, respectively.

FIG. 82 illustrates a variation of FIG. 80, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 and F2 suitable for detection, representing normal transcripte1-e2, and for the alternative splice variant with intron insertioni1-e2, respectively.

FIG. 83 illustrates a close up of low-level alternative splice variant(intron insertion) detection (overview in FIG. 79) using the basicRT-PCR-LDR-qPCR detection protocol with carryover prevention. Theinitial gene-specific reverse-transcription and PCR primers containidentical 8-11 base tails to prevent primer dimers, and are designed toamplify only the minority transcript containing the i1-e2 junction. Thismay be achieved by (i) digesting nucleic acids with pancreatic DNasel,to digest all genomic DNA while leaving the mRNA intact, or (ii) usingprimer sets that span intron i2 as well, e.g., reverse-transcribe frome3, in the presence of blocking primer to i2. Products are detectedusing TaqMan™ probes designed across the ligation junction sequence forthe low-abundant alternative splice variant with intron insertion i1-e2.

FIG. 84 illustrates a variation of FIG. 83, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal F1, representinglow-abundant alternative splice variant with intron insertion i1-e2.

FIG. 85 illustrates a variation of FIG. 83, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal F1 suitable for detection, representing the low-abundantalternative splice variant with intron insertion i1-e2.

When using UniTaq containing LDR probes, they are of the followingformat: upstream probes comprise of a 5′ tag, such as UniTaqAi followedby target-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniTaq Bi′—UniTaq Ci′.

The LDR products may be detected using UniTaq-specific primers of theformat UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai. (where F1 is afluorescent dye that is quenched by Quencher Q). Under these conditions,the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Upstream Exon-Downstream Exon Junction—UniTaqBi′—UniTaq Ci′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye.

For products using three LDR probes to detect transcripts with unknownjunctions, the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Upstream Exon—Bridge sequence—DownstreamExon—UniTaq Bi′—UniTaq Ci′

One of the initial PCR primers or upstream LDR probes may also containan RNA base, 4 additional bases and a blocking group on the 3′ end.RNaseH2 is added to the reaction after the reverse-transcription stepfor the PCR, and/or during the LDR reaction. This assures that notemplate independent products are formed.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (e.g., 94°C. or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstreamprobe may contain a base the same as the 3′ discriminating base on theupstream primer, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

Prophetic Example 4 Accurate Quantification of Tumor-Specific CopyChanges in DNA Isolated from Circulating Tumor Cells

Overview of approach: Since there may be only a few CTC's present in thepurified sample, it is important to use spatial multiplexing toaccurately count every chromosome copy in the sample. This approachdepends on the fidelity of two enzymes: (i) Taq polymerase to faithfullycopy low-level copies of DNA regions in the initial sample, and (ii)ligase in discriminating primers that hybridize adjacent to each other.Once a ligation event has taken place, those products will be amplifiedin a subsequent Real-time PCR amplification step, and thus this is thekey discriminatory step.

To protect against carryover contamination, UNG is added to the reactionprior to polymerase activation, and the initial PCR amplification isperformed with dUTP. The LDR probes are comprised of the natural bases,thus the LDR product is now resistant to UNG digestion in the secondreal-time PCR step. Note that the LDR products contain tags or UniTaqsequences on their non-ligating ends, which are lacking in the targetDNA, thus accidental carryover of LDR products does not result inlarge-scale amplification. Unlike with PCR, an initial LDR product isnot a substrate for a second LDR reaction.

To summarize the levels of discrimination of the above approach usingboth PCR and LDR primers for the determination of copy number detectionof specific regions:

-   1. Use of PCR primers with universal tails, such that if any    target-independent primer dimer formed, the incorrect product will    form a hairpin that will inhibit further amplification.-   2. Use of UNG to prevent carryover contamination of initial PCR    reaction.-   3. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   4. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   5. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed protocol for Highly Accurate Quantification of    Tumor-Specific Copy Changes in DNA or RNA Isolated from Circulating    Tumor Cells.

4.1.a. Incubate genomic DNA in the presence of UNG (37° C., 15-30minutes, to prevent carryover), dUTP, and other dNTP's, AmpliTaq Gold,and gene-specific primers containing universal tails, such that if anytarget-independent primer dimer formed, the incorrect product will forma hairpin that will inhibit further amplification. This initial genomicDNA—PCR reaction mixture is distributed in 12, 24, 48, or 96 individualwells (spatial multiplexing) for multiplex PCR amplification. Denaturegenomic DNA from plasma, inactivate UNG, and activate AmpliTaq Gold (94°C., 5-10 minute) and multiplex PCR amplify chromosomal regions for alimited number of cycles (94° C., 10 sec., 60° C. 30 sec., 72° C. 30sec. for 12-20 cycles). The PCR primers are designed to have Tm valuesaround 64-66° C., and will hybridize robustly, even when used atconcentrations 10 to 50-fold below the norm for uniplex PCR (10 nM to 50nM each primer). The cycles are limited to retain proportional balanceof PCR products with respect to each other, while still amplifying lowabundant sequences about 100,000 to 1,000,000—fold. After PCRamplification, Taq polymerase is inactivated (by incubating at 99° C.for 30 minutes.)

4.1.b. Add thermostable ligase (preferably from strain AK16D), buffersupplement to optimized ligation conditions, and suitable upstream anddownstream LDR probes (10 nM to 20 nM each, downstream probes may besynthesized with 5′ phosphate, or kinased in bulk prior to reactions;Upstream probes comprise of a 5′ tag, such as UniAi followed bytarget-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniCi′. Perform 20 cycles of LDR, (94° C., 10 sec., 60° C. 4-5minutes). This will allow for ligation events to occur on the PCRproducts if chromosomal DNA is present.

4.1.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR primers would be UniAi and UniCi respectively,and the products would be of the form:

UniAi—Chromosomal Target Region—UniCi′

FIG. 86 illustrates DNA copy number enumeration with spatialmultiplexing (see FIGS. 6-9), using the basic PCR-LDR-q PCR detectionprotocol with carryover prevention. The initial gene-specific PCRprimers contain identical 8-11 base tails to prevent primer dimers.Products are detected using TaqMan™ probes designed across the ligationjunction sequence for each chromosomal region, and total copy numberenumerated.

FIG. 87 illustrates a variation of FIG. 86, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal for eachchromosomal region, and total copy number enumerated.

FIG. 88 illustrates a variation of FIG. 86, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support(see FIGS. 19-23). The LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection of eachchromosomal region, and total copy number enumerated.

FIG. 89 illustrates mRNA transcript number enumeration with spatialmultiplexing (see FIGS. 10-13), using the basic reverse-transcriptionPCR-LDR-qPCR detection protocol with carryover prevention. The initialgene-specific reverse-transcription and PCR primers contain identical8-11 base tails to prevent primer dimers. Products are detected usingTaqMan™ probes designed across the ligation junction sequence for eachmRNA transcript, to enable accurate enumeration.

FIG. 90 illustrates a variation of FIG. 89, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal for each mRNAtranscript, to enable accurate enumeration.

FIG. 91 illustrates a variation of FIG. 89, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support(see FIGS. 24-28). The LDR probes are designed to contain shortcomplementary sequences that only hybridize to each other when ligatedtogether, generating FRET signal suitable for detection of each mRNAtranscript, to enable accurate enumeration.

As an example of spatial multiplexing across 48 wells, consider DNAisolated from 12 CTC's, with probes prepared for various copy regionswith prognostic or therapeutic value, such as loss of heterozygosity ofchromosomal arm 8p (LOH at 8p; predicts worse outcome), or amplificationof the Her2 gene at 17q12 (predicts responsiveness to Herceptintherapy). Multiple LDR probe sets may be employed to determine copynumber across the genome, with additional pairs at focal points known toundergo significant amplification. For this example, the diploid regionsof the genome would produce signal from 24 copies (i.e. 2×12 cells), anLOH event at 8p would produce 12 copies, and for example if the Her2gene was amplified 8-fold, it would produce signal from 96 copies (i.e.8×12). The likely Poisson distribution (FIGS. 31 & 32) show the LOHregion will have a distribution of about (wells: molecules) (38:0; 10:1;1:2) for 12 molecules, the diploid regions will have a distribution ofabout (29:0; 15:1; 4:2; 1:3) for 24 molecules, while the amplifiedregion will have a distribution of about (6:0; 13:1; 13:2; 9:3; 4:4;2:5; 1:6) for 96 molecules. Note that even if a region has undergoneonly mild amplification, for example from 2 copies per cell to 3 copies,trisomy regions will have a distribution of about (23:0; 17:1; 6:2; 2:3)for 36 molecules, and thus distinguishable from diploid regions. Asbefore, even if LDR-TaqMan™ LDR signal is variable enough thatdistinguishing the higher level signals becomes too difficult, as longas the signal is clean enough to distinguish 0, 1, and 2 initialmolecules (represented as LDR-TaqMan™ Ct values of 12.5, 10, and 9, orLDR signals of about 1,000, 5,000, and 10,000, respectively), thisapproach will have no difficulty distinguishing and enumerating regionsthat have undergone LOH, regions that are diploid, and regions that haveundergone amplification. The CT or fluorescent signal is variable enoughonly to distinguish between 0 and 1 or more initial chromosomalmolecules, given 24 copies of diploid chromosomes in a minimum of 48wells, this approach should distinguish and enumerate regions that haveundergone LOH, regions that are diploid, and regions that have undergoneamplification.

For cfDNA samples with higher tumor DNA load, or with mRNA or miRNA,where the starting target is present in higher amounts, the LDR signalwill be proportionally stronger. A large dynamic range of initialmolecules may also be achieved by using a different strategy fordiluting the initial signal. For example, after a reverse-transcriptionstep for mRNA isolated from exosomes, instead of dividing the sampleequally among 48 wells, the sample is distributed into 10 aliquots, thefirst 8 are distributed into wells, and one of the remaining aliquots isdiluted into10 aliquots, with 8 distributed into wells, etc. This allowsfor 6 orders of magnitude of dilution: (i.e. 8×6=48). Examination ofPoisson distributions shows that as long as 1 well in the last dilutionrepresents 0 molecules, a given set of 8 wells can provide asemi-quantitative estimate of starting molecules over a 2 order ofmagnitude dynamic range, from 1 to 128 molecules, even while the LDRreadout only needs to provide a 20-fold dynamic range, or Ct range of4-5 (see FIGS. 33-37 that show Poisson distribution of from 1 to 128molecules in 8 wells). Since the dilutions are only 10-fold, at least 2sets of 8 wells may be used to determine the number of originalmolecules of multiple different transcripts in the sample, even if somemRNA molecules were on the order of 10 molecules, while others were onthe order of 1×10⁶ molecules.

The same approach may be used for quantifying RNA copy, but reversetranscriptase is added in the first step, and the spatial distributionof initial reaction mix may be dilution & distribution as outlinedabove.

When using UniTaq containing LDR probes, they are of the followingformat: upstream probes comprise of a 5′ tag, such as UniTaqAi followedby target-specific sequence with a C:A or G:T mismatch at the 3^(rd) orpenultimate base, the mutation base at the 3′ end, followed by an RNAbase and 4 more DNA bases that matches the target, and a C3spacer-blocking group. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniTaq Bi′—UniCi′.

The LDR products may be detected using UniTaq-specific primers of theformat UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai. (where F1 is afluorescent dye that is quenched by Quencher Q). Under these conditions,the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Chromosomal Target Region—UniTaq Bi′—UniTaq Ci′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye.

One of the initial PCR primers (with RNA) or both initial PCR primers(with DNA) or upstream LDR probes may also contain an RNA base, 4additional bases and a blocking group on the 3′ end. When quantifyingDNA copy, RNaseH2 is added with the PCR reaction, and/or with the LDRreaction. When quantifying RNA, RNaseH2 is added to the reaction afterthe reverse-transcription step with the PCR, and/or with the LDRreaction. This assures that no template independent products are formed.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (e.g. 94° C.or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstreamprimer may contain a base the same as the 3′ discriminating base on theupstream primer, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

Prophetic Example 5 Accurate Quantification of miRNA, lncRNA, or mRNAChanges from Isolated Exosomes, or from Circulating Tumor Cells

Overview of approach: This approach depends on the fidelity of twoenzymes: (i) Reverse Transcriptase and Taq polymerase to faithfully copylow-level copies of miRNA in the initial sample, and (ii) the ligase indiscriminating probes hybridized adjacent to each other. Once a ligationevent has taken place, those products will be amplified in a subsequentReal-time PCR amplification step, and thus this is the keydiscriminatory step.

MicroRNA (miRNA) have been identified as potential tissue-specificmarkers of the presence of tumors, their classification andprognostication. miRNA exist in serum and plasma either as complexeswith Ago2 proteins or by encapsulation as exosomes.

To protect against carryover contamination, UNG is added to the reactionprior to reverse transcription by MMLV, and the initial PCRamplification is performed with dUTP. The LDR probes are comprised ofthe natural bases, thus the LDR product is now resistant to UNGdigestion in the second real-time PCR step. Note that the LDR productscontain tags or UniTaq sequences on their non-ligating ends, which arelacking in the target miRNA, thus accidental carryover of LDR productsdoes not result in large-scale amplification. Unlike with PCR, aninitial LDR product is not a substrate for a second LDR reaction.

A miRNA specific hairpin loop containing a universal reverse primerregion and an 6-8 base miRNA specific region is hybridized to the 3′ endof the miRNA and extended with MMLV in the presence of dUTP. Under theright conditions, MMLV will add 2-3 C nucleotides past the 5′ end of themiRNA template in a template-independent extension reaction.

After the initial cDNA generation, add a universal reverse primer anduniversal-tailed forward primer that hybridize to the 2-3 additional Cnucleotides and from 12 to 14 specific bases of the miRNA. Taqpolymerase is used to perform 16-20 cycles of universal amplification;the universal reverse primer is located to eliminate most of the hairpinregion during the cDNA generation.

To summarize the levels of discrimination of the above approach for highsensitivity detection of miRNA:

-   1. Use of UNG to prevent carryover contamination of initial reverse    transcription and PCR reactions.-   2. Use of 3′ ligation fidelity of thermostable ligase on upstream    LDR probe.-   3. Use of UniTaq or tag primers to amplify LDR products for    real-time PCR readout.-   4. Use of UNG to prevent carryover contamination of real-time PCR    reaction.    Detailed Protocol for Highly Sensitive Detection of miRNA:

5.1.a. Incubate isolated miRNA (or even total isolated nucleic acids) inthe presence of UNG (37° C., 15-30 minutes, to prevent carryover), dUTP,and other dNTP's, MMLV reverse transcriptase, AmpliTaq Gold, andtranscript-specific primers. This initial cDNA—PCR reaction mixture issuitable for multiplex reverse-transcription PCR amplification in 12,24, 48, or 96 individual wells (spatial multiplexing), or in a singlewell. After extension of hairpin reverse primers on their cognate miRNAto generate cDNA, inactivate UNG and MMLV reverse transcriptase, andactivate AmpliTaq Gold (94° C., 5-10 minute) and multiplex PCR amplifytranscript-containing fragments using bridge and tag primers Ti, and Tjfor a limited number of cycles (94° C., 10 sec., 60° C. 30 sec., 72° C.30 sec. for 16-20 cycles). After PCR amplification, Taq polymerase isinactivated (by incubating at 99° C. for 30 minutes.)

5.1.b. Add thermostable ligase (preferably from strain AK16D), buffersupplement to optimized ligation conditions, and suitable upstream anddownstream LDR probes (10 nM to 20 nM each, downstream probes may besynthesized with 5′ phosphate, or kinased in bulk prior to reactions;Upstream probes comprise of a 5′ tag, such as UniAi followed bytarget-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniCi′. Perform 20 cycles of LDR, (94° C., 10 sec., 60° C. 4-5minutes). This will allow for ligation events to occur on the PCRproducts if chromosomal DNA is present.

5.1.c. Open tube/wells, dilute (10- to 100-fold) and distribute aliquotsto wells for Real-Time PCR reactions, each well containing theappropriate TaqMan™ master mix with UNG for carryover prevention, andthe following primers: UniCi and UniAi, and a TaqMan™ probe that coversthe sequence across the ligation junction. Under such conditions, thetag sequences on the LDR probes would be UniAi and UniCi respectively,and the products would be of the form:

UniAi—Upstream miRNA—Downstream miRNA Junction—UniCi′

In one variation of the above theme, the hairpin oligonucleotide isligated to the 3′ end of the miRNA in a base-specific manner, appendingan artificial loop sequence, which contains a tag-primer binding site(Tj). This enables extension to copy the entire miRNA sequence, as wellas initiating a PCR reaction using miRNA target-specific bridge primers(comprising of (Ti) and miRNA-specific sequence), and the two tag primer(Ti, Tj). The PCR product is now suitable for subsequent LDR step asdescribed above.

FIG. 92 illustrates miRNA detection, using reverse transcription of aloop primer, followed by tag and bridge primer PCR protocol withcarryover prevention. Subsequent LDR-q PCR detection protocol is usedwith carryover prevention. The initial miRNA-specific PCR primerscontain identical 8-11 base tails as part of the tag primers to preventprimer dimers. Products are detected using TaqMan™ probes designedacross the ligation junction sequence for each miRNA detected.

FIG. 93 illustrates a variation of FIG. 92, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal for each miRNA.

FIG. 94 illustrates a variation of FIG. 92, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal suitable for detection of each miRNA.

FIG. 95 illustrates miRNA detection, using ligation of a loop primerdirectly to miRNA, followed reverse transcription using a tag primercomplementary to the loop with carryover prevention. The procedure usesa tag and bridge primer for PCR-LDR-q PCR detection, with carryoverprevention. The initial miRNA-specific PCR primers contain identical8-11 base tails as part of the tag primers to prevent primer dimers.Products are detected using TaqMan™ probes designed across the ligationjunction sequence for each miRNA detected.

FIG. 96 illustrates a variation of FIG. 95, where upstream anddownstream LDR primers contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal for each miRNA.

FIG. 97 illustrates a variation of FIG. 95, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal suitable for detection of each miRNA.

FIG. 98 illustrates a variation of FIG. 95, where the bridge PCR primercontains an RNA base, 4 additional bases and a blocking group on the 3′end. RNaseH2 is added to the reaction after the reverse-transcriptionstep to unblock the PCR primer. This assures that no templateindependent products are formed.

FIG. 99 illustrates a variation of FIG. 98, where upstream anddownstream LDR probes contain UniTaq Ai and UniTaq Bi′-UniTaq Ci′ tagsrespectively, and products are detected using fluorescently labeledUniTaq primer F1-UniTaq Bi—Q—UniTaq Ai to detect signal for each miRNA.

FIG. 100 illustrates a variation of FIG. 98, where the PCR products aredistributed (spatial multiplexing), and are captured on a solid support.The LDR probes are designed to contain short complementary sequencesthat only hybridize to each other when ligated together, generating FRETsignal suitable for detection of each miRNA.

When using UniTaq containing LDR probes, they are of the followingformat: upstream probes comprise of a 5′ tag, such as UniTaqAi followedby target-specific sequence. The downstream probes comprise a 5′phosphorylated end, followed by target-specific sequence, and a 3′ tag,such as UniTaq Bi′—UniTaq Ci′.

The LDR products may be detected using UniTaq-specific primers of theformat UniTaq Ci and F1-UniTaq Bi—Q—UniTaq Ai. (where F1 is afluorescent dye that is quenched by Quencher Q). Under these conditions,the following product will form: F1-UniTaq Bi—Q—UniTaq Ai—UpstreammiRNA—Downstream miRNA Junction—UniTaq Bi′—UniTaq Ci′

This construct will hairpin, such that the UniTaq Bi sequence pairs withthe UniTaq Bi′ sequence. When UniTaq Primer Ci binds to the UniTaq Ci′sequence, the 5′→3′ exonuclease activity of polymerase digests theUniTaq Bi sequence, liberating the F1 fluorescent dye.

For products using three LDR probes to detect transcripts with unknownjunctions, the following product will form:

F1-UniTaq Bi—Q—UniTaq Ai—Upstream Exon—Bridge Sequence—DownstreamExon—UniTaq Bi′—UniTaq Ci′

One of the initial PCR primers or upstream LDR probes may also containan RNA base, 4 additional bases and a blocking group on the 3′ end.RNaseH2 is added to the reaction after the reverse-transcription stepfor the PCR, and/or during the LDR reaction. This assures that notemplate independent products are formed.

The downstream LDR probes may also be phosphorylated during the ligationreaction using thermophilic phage kinase (derived from bacteriophageRM378 that infects Rhodothermus marinus). Under these conditions thedenaturation step in the LDR should be as short as possible (e.g., 94°C. or even lower for 1 second), as the thermophilic kinase is not fullythermostable—or just preincubate at 65° C. for 15 minute to achieve fullprimer phosphorylation. Alternatively, the 5′ side of the downstreamprobe may contain a base the same as the 3′ discriminating base on theupstream probe, said base removed by the 5′ to 3′ nuclease activity ofFen nuclease or Taq polymerase to liberate a 5′ phosphate suitable for asubsequent ligation.

Empirical Examples Detection of Cancer-Related Mutations and Methylationby PCR-LDR-qPCR

General Methods for Empirical Examples 1-4.

The cell lines used were: HT-29 colon adenocarcinoma cell line, whichharbors the V600E (1799T>A) BRAF heterozygotic mutation; HEC-1 (A)endometrium adenocarcinoma cell line, which harbors the R248Q (743G>A)TP53 heterozygotic mutation and the G12D (35G>A) KRAS heterozygoticmutation; LS123 colon adenocarcinoma cell line, which harbors the G12S(34G>A) KRAS heterozygotic mutation; SW1463 colon adenocarcinoma cellline, which harbors the G12C (34G>T) KRAS homozygotic mutation; SW480colon adenocarcinoma cell line, which harbors the G12V (35G>T) KRAShomozygotic mutation; and SW1116 colon adenocarcinoma cell line, whichharbors the G12A (35G>C) KRAS heterozygotic mutation. All cell lineswere seeded in 60 cm² culture dishes in McCoy's 5a medium containing 4.5g/1 glucose, supplemented with 10% fetal calf serum and kept in ahumidified atmosphere containing 5% CO₂. Once cells reached 80-90%confluence they were washed in Phosphate Buffered Saline (×3) andcollected by centrifugation (500×g). DNA was isolated using the DNeasyBlood & Tissue Kit (Qiagen; Valencia, Calif.). DNA concentration wasdetermined with Quant-iT Picogreen Assay LifeTechnologies/ThermoFisher;Waltham, Mass.). Human Genomic DNA (0.2 mg/ml)containing high molecμlar weight (>50 kb) genomic DNA isolated fromhuman blood (buffy coat) (Roche hgDNA) was purchased from Roche(Indianapolis, Ind.). Its accurate concentration was determined to be 39ng/μl by Quant-iT PicoGreen dsDNA Assay Kit.

Human plasma (with K2 EDTA as an anti-coagμlant) from cancer-freedonors, 21-61 years of age was purchased from BioreclamationIVT (Nassau,N.Y.). DNA was isolated from individual plasma samples (5 mL) using theQIAamp Circμlating Nucleic Acid Kit according to manufacturer'sinstructions, and quantified with Quant-iT Picogreen Assay LifeTechnologies/ThermoFisher; Waltham, Mass.).

Empirical Example 1 Detection of V600E (1799T>A) BRAF Mutation

All primers used are listed in Table 1. All primers were purchased fromIntegrated DNA Technologies Inc. (IDT, Coralville, Iowa), except forprimer iCDx-315-BRAF_FLW, which was purchased from Exiqon Inc. (Woburn,Mass.).

TABLE 1  Primers for PCR-LDR-qPCR detection of BRAF V600E mutationPrimer Name Step Primer Sequence iCDx-328-Braf_ PCRCCTCACAGTAAAAATAGGTGATTTT PF_WT_blk2 GGTCTArGCTAT/3SpC3/  (SEQ ID NO: 1)iCDx-284-Br600-PR PCR GGTGTCGTGGTCAAAATGGATCCAG ACAACTGTTCAAAC (SEQ ID NO: 2) iCDx-315-BRAF_FLW PCR /5Phos/GCTA+C+AG+T+G-FAAAT+CTCG/3SpC3/ (SEQ ID NO: 3) iCDx-308-Br600_ LDRTAGCGATAGTACCGACAGTCACGTCCTA (3)-L_Up_Rm AATAGGTGATTTTGGTCTAGCTACGGArGAAAC/3SPC3/ (SEQ ID NO: 4) iCDx-276-Br600- LDR/5Phos/GAAATCTCGATGGAGTGGGT L_Dn_P CCCATTTGGTGTGCGGAAACCTATCGTCGA (SEQ ID NO: 5) iCDx-277_A4 qPCR TAGCGATAGTACCGACAGTCAC (SEQ ID NO: 6) iCDx-279_C4 qPCR TCGACGATAGGTTTCCGCAC  (SEQ ID NO: 7)iCDx-281-Br600_ qPCR 5′-/56-TAMN/TA CGG AGA AAT (3)_ProbeCTC GAT GGA GTG GGT/ 3IAbRQSp/-3′ (SEQ ID NO: 8) /5SpC3/-5′ C3 Spacer,/3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ Fam FlourescentDye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ Flourescent Quencher ™,/3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green to pink spectralrange, “+”-Locked Nucleic Acid base, “rA”-ribonucleotide baseriboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

Dilution experiments. The PCR step was performed in a 10 μl reactionprepared by adding: 1.58 μl of nuclease free water (IDT), 2 μl of GotaqFlexi buffer 5× without Magnesium (Promega, Madison, Wis.), 0.8 μl ofMgCl₂ at 25 mM (Promega, Madison, Wis.), 0.2 μl of dNTPs (with dATP,dCTP, dGTP and dUTP, 10 mM each) (Promega, Madison, Wis.), 0.25 μl ofiCDx-328-Braf PF WT blk2 forward primer at 2 μM, 0.25 μl ofiCDx-284-Br600-PR reverse primer at 2 μM, 1.25 μl of iCDx-284-Br600-PRLNA blocking primer at 2 μM, 0.25 μl of RNAseH2 (IDT) at 20 mU/μl(diluted in RNAseH2 dilution buffer from IDT), 0.2 μl of Antarcticthermolabile UDG (New England Biolabs (NEB), Ipswich, Mass.) at 1 U/μl,and 0.22 μl of Klentaql polymerase (DNA Polymerase Technology, St.Louis, Mo.) mixed with Platinum Taq Antibody (Invitrogen/ThermoFisherWaltham, Mass.) (the mix is prepared by adding 0.02 μl of Klentaqlpolymerase at 50 U/μl to 0.2 μl of Platinum Taq Antibody at 5 U/μl), and3 μl of corresponding template. Templates were: nuclease free water forthe NTC—Non Template Control—, Roche hgDNA at 11.7 ng/μl (thus, 35 ng or10000 Genome Equivalents (GE) in 3 μl)—wild type—, and HT-29 wcDNA mixedwith Roche hgDNA as follows: 1) 0.047 ng/μl of wcDNA HT-29 in 11.7 ng/μlof Roche hgDNA, thus 0.14 ng of wcDNA HT-29 and 35 ng of Roche hgDNA in3 μl, which corresponds to 40 GE HT-29 (only 20 GE are mutated) and10000 GE of Roche human genomic DNA (i.e. 1 mutant (mt) in 500 wild type(wt)); 2) 0.023 ng/μl of wcDNA HT-29 in 11.7 ng/μl of Roche hgDNA, thus0.07 ng of wcDNA HT-29 and 35 ng of Roche hgDNA in 3 μl, whichcorresponds to 20 GE HT-29 (only 10 GE are mutated) and 10000 GE ofRoche hgDNA; (i.e. 1 mt in 1000 wt) 3) 0.0117 ng/μl of wcDNA HT-29 in11.7 ng/μl of Roche hgDNA, thus 0.0035 ng of wcDNA HT-29 and 35 ng ofRoche hgDNA in 3 μl, which corresponds to 10 GE HT-29 (only 5 GE aremutated) and 10000 GE of Roche hgDNA (i.e. 1 mt in 2000 wt); 4) 0.006ng/μl of wcDNA HT-29 in 11.7 ng/μl of Roche hgDNA, thus 0.00175 ng ofwcDNA HT-29 and 35 ng of Roche hgDNA in 3 μl, which corresponds to 5 GEHT-29 (only 2 or 3 GE are mutated) and 10000 GE of Roche hgDNA (i.e. 1mt in 5000 wt). Note: after preparing the 0.047 ng/μl of wcDNA HT-29 in11.7 ng/μl of Roche hgDNA mix (40 GE of mutant in 10000 GE of RochehgDNA), rest of mutant plus Roche hgDNA mixes are prepared by serialdilutions mixing 0.5 volumes of the preceding mutant-Roche hgDNA mixplus 0.5 volumes of Roche hgDNA at 11.7 ng/μl, so the mutant GE arediluted ½ while Roche hgDNA GE remains undiluted (10000 GE). PCRreactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler (Applied Biosystems/ThermoFisher; Waltham, Mass.) and thefollowing program: 30 min at 37° C., 2 min at 95° C., 40 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C.

The LDR step was performed in a 10 μl reaction prepared by adding: 5.82μl of nuclease free water (IDT), 1 μl of 10× AK16D ligase reactionbuffer [1× buffer contains 20 mM Tris-HCl pH 8.5 (Bio-Rad, Hercules,Calif.), 5 mM MgCl₂ (Sigma-Aldrich, St. Louis, Mo.), 50 mM KCl(Sigma-Aldrich, St. Louis, Mo.), 10 mM DTT (Sigma-Aldrich, St. Louis,Mo.) and 20 ug/ml of BSA (Sigma-Aldrich, St. Louis, Mo.)], 0.25 μl ofDTT (Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl ofNAD⁺(Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of RNAseH2 (IDT)at 20 mU/μ1, 0.2 μl of iCDx-308-Br600_(3)-L_Up_Rm Up primer at 500 nM,0.2 μl of iCDx-276-Br600-L_Dn_P Down primer at 500 nM, 0.028 μl ofpurified AK16D ligase at 8.8 μM, and 2 μl of PCR reaction. LDR reactionswere run in BioExcell Clear 96 Well PCR 0.2 ml plates (Worldwide MedicalProducts, Inc., Bristol, Pa.) sealed with MicroAmp® clear adhesive filmfrom Applied Biosystems (Applied Biosystems/ThermoFisher; Waltham,Mass.), using a Proflex PCR system thermocycler (AppliedBiosystems/ThermoFisher; Waltham, Mass.) and the following program: 20cycles of (10 sec at 94° C., and 4 min at 60° C.) followed by a finalhold at 4° C.

The qPCR step was performed in a 10 μl reaction prepared by adding: 1.5μl of nuclease free water (IDT), 5 μl of 2× TaqMan® Fast Universal PCRMaster Mix (fast amplitaq, UDG and dUTP) from Applied Biosystems(Applied Biosystems/ThermoFisher; Waltham, Mass.), 1 μl of iCDx-277 A4forward primer at 2.5 μM, 1 μl of iCDx-279_C4 reverse primer at 2.5 μM,0.5 μl of iCDx-281-Br600_(3)_Probe Taqman probe at 5 μM, and 1 μl of LDRreaction. qPCR reactions were run in a ViiA7 real-time thermocycler fromApplied Biosystems (Applied Biosystems/ThermoFisher; Waltham, Mass.),using MicroAmp® Fast-96-Well Reaction 0.1 ml plates sealed withMicroAmp™ Optical adhesive film Applied Biosystems/ThermoFisher;Waltham, Ma, and the following setting: fast block, Standard curve asexperiment type, ROX as passive reference, Ct as quantification method(automatic threshold, but adjusted to 0.04 when needed), TAMRA asreporter, and NFQ-MGB as quencher; and using the following program: 2min at 50° C., and 40 cycles of (1 sec at 95° C., and 20 sec at 60° C.).Results are shown in FIG. 159 and Table 2.

TABLE 2 Results of dilution experiments to detect BRAF V600E. CtDifference Starting Genome vs Equivalents 10000 GE of Templates (per 10μl of PCR) Ct Roche hgDNA HT-29_1 (BRAF 40 (20 mt) + 16.1 16.7 V600E,het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 500 wt) HT-29_1 (BRAF 20(10 mt) + 22.4 10.4 V600E, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT)1,000 wt) HT-29_1 (BRAF 10 (5 mt) + 22.7 10.2 V600E, het) + 10,000 wt(i.e. 1 mt/ Roche hgDNA (WT) 2,000 wt) HT-29_1 (BRAF 5 (2 or 3 mt) +22.0 10.9 V600E, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 5,000 wt)Roche hgDNA (WT) 10,000 wt 32.8 NTC N/A Undetermined Mt = mutant BRAFV600E. WT = wild-type BRAF. Het = heterozygote. UD = Undetermined. Ctvalues in bold correspond to Cts obtained from “true curves”, and valuesin normal font type correspond to values obtained from “flat curves,”i.e. non-baseline curves that appear in the late amplification cycles(generally after a Ct value of 36).

Pixel experiments using hgDNA from Roche. The PCR step was performed ina 130 μl mixture prepared by adding: 56.54 μl of nuclease free water(IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium (Promega,Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison, Wis.), 2.6μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each) (Promega,Madison, Wis.), 3.25 μl of iCDx-328-Braf PF WT blk2 forward primer at 2μM, 3.25 μl of iCDx-284-Br600-PR reverse primer at 2 μM, 16.25 μl ofiCDx-284-Br600-PR LNA blocking primer at 2 μM, 3.25 μl of RNAseH2 (IDT)at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT), 2.6 μl ofAntarctic thermolabile UDG (New England Biolabs (NEB), Ipswich, Mass.)at 1 U/μl, and 2.86 μl of Klentaql polymerase (DNA PolymeraseTechnology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μ1 to 3 μl of Platinum Taq Antibody at 5U/μl), and 3 μl of corresponding template. Templates were: nuclease freewater for the NTC—Non Template Control —, Roche hgDNA at 2.925 ng/μl(thus, 8.75 ng of Roche hgDNA or 2500 Genome Equivalents (GE) in 3μl)—wild type—, and HT-29 wcDNA mixed with Roche hgDNA as follows: 1)0.023 ng/μl of wcDNA HT-29 in 2.925 ng/μl of Roche hgDNA, thus 0.07 ngof wcDNA HT-29 and 8.75 ng of Roche hgDNA in 3 μl, which corresponds to20 GE HT-29 (only 10 GE are mutated) and 2500 GE of Roche hgDNA; 2)0.0117 ng/μl of wcDNA HT-29 in 2.925 ng/μl of Roche hgDNA, thus 0.0035ng of wcDNA HT-29 and 8.75 ng of Roche hgDNA in 3 μl, which correspondsto 10 GE HT-29 (only 5 GE are mutated) and 2500 GE of Roche hgDNA. Note:the 0.0117 ng/μl of wcDNA HT-29 in 2.925 ng/μl of Roche hgDNA mix isprepared by serial dilution mixing 0.5 volume of the 0.023 ng/μl ofwcDNA HT-29 in 2.925 ng/μl of Roche hgDNA mix (20 GE of mutant, 10 GEmutated, in 2500 GE of Roche hgDNA) plus 0.5 volume of Roche hgDNA at2.925 ng/μl, so the mutant is diluted to 10 GE (5 GE mutated) whileRoche hgDNA GE remains undiluted (2500 GE).

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Mass. and thefollowing program: 30 min at 37° C., 2 min at 95° C., 40 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section.Results are shown in FIG. 160 and Table 3.

TABLE 3 Results of pixel experiments to detect BRAF V600E diluted inRoche hgDNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 9 1011 12 Amplifications HT-29_1 (BRAF 20 GE (10 mt) 27.7 23.0 25.2 27.527.4 26.0 35.7 22.6 24.9 27.1 25.8 26.4 11 V600E, het) + + Roche hgDNA(wt) 2500 GE (wt) HT-29_1 (BRAF 10 GE (5 mt) 35.8 31.8 27.5 36.0 26.531.1 31.3 23.1 35.9 23.7 36.3 35.7 7 V600E, het )+ + Roche hgDNA (wt)2500 GE (wt) Roche hgDNA (wt) 2500 GE 36.5 37.5 36.3 36.4 35.8 37.2 36.036.9 37.0 36.8 36.8 36.0 0 (wt) NTC N/A UD 39.8 39.1 38.1 UD UD 39.5 UD39.1 35.5 UD 38.8 0 Mt = mutant BRAF V600E. wt = wild-type BRAF. Het =heterozygote. UD = Undetermined. Columns 1-12 correspond to the Ctsobtained from tubes 1-12, respectively, with values in boldcorresponding to Cts obtained from “true curves”, and values in normalfont type corresponding to values obtained from “flat curves,” i.e.non-baseline curves that appear in the late amplification cycles(generally after a Ct value of 36).

Pixel experiments using human plasma DNA (plasma #8). The PCR step wasperformed in a 130 μl mixture prepared by adding: 46.54 μl of nucleasefree water (IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium(Promega, Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison,Wis.), 2.6 μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each)(Promega, Madison, Wis.), 3.25 μl of iCDx-328-Braf_PF_WT_blk2 forwardprimer at 2 μM, 3.25 μl of iCDx-284-Br600-PR reverse primer at 2 μM,16.25 μl of iCDx-284-Br600-PR LNA blocking primer at 2 μM, 3.25 μl ofRNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT),2.6 μl of Antarctic thermolabile UDG (New England Biolabs (NEB),Ipswich, Mass.) at 1 U/μl, and 2.86 μl of Klentaql polymerase (DNAPolymerase Technology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μl to 3 μl of Platinum Taq Antibody at 5U/μl), and 13 μl of corresponding template. Templates were: nucleasefree water for the NTC—Non Template Control—, Plasma DNA (prepared as6.9 μl nuclease free H₂O plus 6.1 μl of plasma DNA at 0.714 ng/μl thus,4.375 ng of plasma DNA or 1250 Genome Equivalents (GE) in the PCRreaction)—wild type—and HT-29 wcDNA mixed with Plasma DNA as follows: 1)4.9 μl nuclease free H2O, plus 6.1 μl, Plasma DNA at 0.714 ng/μl, plus 2μl of 0.035 ng/μl of wcDNA HT-29, thus, 4.375 ng of plasma DNA or 1250Genome Equivalents (GE) plus 0.07 ng of wcDNA HT-29 which corresponds to20 GE HT-29 (only 10 GE are mutated) in the PCR reaction; 2) 5.9 μlnuclease free H2O, plus 6.1 μl Plasma DNA at 0.714 ng/μl, plus 1 μl of0.035 ng/μl of wcDNA HT-29, thus, 4.375 ng of plasma DNA or 1250 GenomeEquivalents (GE) plus 0.035 ng of wcDNA HT-29 which corresponds to 10 GEHT-29 (only 5 GE are mutated) in the PCR reaction. Note: The mix with4.375 ng of plasma DNA or 1250 Genome Equivalents (GE) plus 0.035 ng ofwcDNA (10 GE of mutant, 5 mutated) is prepared by serial dilution mixing0.5 volumes of the previous mix with 4.375 ng of plasma DNA or 1250Genome Equivalents (GE) and 0.07 ng of wcDNA (20 GE of mutant, 10mutated) with 0.5 volumes of the 4.375 ng of plasma DNA mix.

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Mass. and thefollowing program: 30 min at 37° C., 2 min at 95° C., 45 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section.Results are shown in FIG. 161 and Table 4.

TABLE 4 Results of pixel experiments to detect BRAF V600E diluted inhuman plasma DNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 910 11 12 Amplifications HT-29_1 (BRAF 20 GE (10 mt) 35.7 22.4 19.4 34.321.1 19.1 28.2 16.4 34.4 29.1 34.7 18.0 8 V600E, het) + + Human plasma1250 GE DNA (wt) (wt) HT-29_1 (BRAF 10 GE (5 mt) 20.3 35.5 16.6 35.034.8 19.3 34.3 34.2 35.3 34.3 36.0 34.9 3 V600E, het) + + Human plasma1250 GE DNA (wt) (wt) Human plasma 1250 GE 36.4 UD 25.6 36.0 35.1 35.734.7 35.3 35.8 36.2 36.2 33.0 1 DNA (wt) (wt) NTC N/A UD 39.9 UD UD 38.2UD UD UD UD UD UD UD 0 Mt = mutant BRAF V600E. wt = wild-type BRAF. Het= heterozygote. UD = Undetermined. Columns 1-12 correspond to the Ctsobtained from tubes 1-12, respectively, with values in boldcorresponding to Cts obtained from “true curves”, and values in normalfont type corresponding to values obtained from “flat curves,” i.e.non-baseline curves that appear in the late amplification cycles(generally after a Ct value of 36).

Empirical Example 2 Detection of R248Q (743G>A) TP53 Mutation

All primers used are listed in Table 5. All primers were purchased fromIntegrated DNA Technologies Inc. ((IDT), Coralville, Iowa), except forPNA primers, which were purchased from PNABio Inc. (Thousand Oaks,Calif.).

TABLE 5  Primers for PCR-LDR-qPCR detection of TP53 R248Q mutationPrimer Name Step Primer Sequence iCDx-326-p53- PCRCCTGCATGGGCGGCATGrAACCG/3SpC3/ 248_PF_WT_blk2 (SEQ ID NO: 9)iCDx-248-p53- PCR GGTGTCGTGGAAGTGGCAAGTGGCTCC 248_PRTGAC (SEQ ID NO: 10) PNA-p53-248-10 PCR GAACCGGAGG (SEQ ID NO: 11)PNA-p53-248-11L PCR TGAACCGGAGG (SEQ ID NO: 12) iCDx-305-P53- LDRTCACTATCGGCGTAGTCACCACAGACGCA 248(3)-L_Up_RmTGGGCGGCATGAATCArGAGGT/3SPC3/ (SEQ ID NO: 13) iCDx-202-P53- LDR/5Phos/GAGGCCCATCCTCACCATCATCA 248-L_Dn_P CGTTGTTGGTGACTTTACCCGGAGGA(SEQ ID NO: 14) iCDx-82_GTT- qPCR TCACTATCGGCGTAGTCACCA (SEQ ID GCGC_A2NO: 15) iCDx-244-C2 qPCR TCCTCCGGGTAAAGTCACCA (SEQ ID NO: 16)iCDx-228-p53- qPCR 5′-/56-FAM/CG GCA TGA A/ZEN/T  248_Probe_sCAG AGG CCC ATC C/3IABkFQ/  (SEQ ID NO: 17) PNA = Peptide nucleic acid,/5SpC3/-5′ C3 Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate,/56-FAM/-5′ Fam Flourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN™ Flourescent Quencher ™, /3IABkFQ/-3′ Iowa Black ® FlourescentQuencher, green to pink spectral range, “+”-Locked Nucleic Acid base,“rA”-ribonucleotide base riboadenosine; “rT”-ribonucleotide baseribothymidine; “rG”-ribonucleotide base riboguanosine;“rC”-ribonucleotide base ribocytosine

Dilution experiments. The PCR step was performed in a 10 μl reactionprepared by adding: 1.58 μl of nuclease free water (IDT), 2 μl of GotaqFlexi buffer 5× without Magnesium (Promega, Madison, Wis.), 0.8 μl ofMgCl₂ at 25 mM (Promega, Madison, Wis.), 0.2 μl of dNTPs (with dATP,dCTP, dGTP and dUTP, 10 mM each) (Promega, Madison, Wis.), 0.25 μl ofiCDx-326-p53-248_PF_WT_blk2 forward primer at 2 μM, 0.25 μl ofiCDx-248-p53-248_PR reverse primer at 2 μM, 1.25 μl of PNA-p53-248-10PNA blocking primer (or PNA-p53-248-11L in the latest experiments) at 2μM, 0.25 μl of RNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilutionbuffer from IDT), 0.2 μl of Antarctic thermolabile UDG (New EnglandBiolabs (NEB), Ipswich, Mass.) at 1 U/μl, and 0.22 μl of Klentaqlpolymerase (DNA Polymerase Technology, St. Louis, Mo.) mixed withPlatinum Taq Antibody (Invitrogen, Carlsbad, Calif.) (the mix isprepared by adding 0.02 μl of Klentaql polymerase (stock at 50 U/μl) to0.2 μl of Platinum Taq Antibody (stock at 5 U/μl), and 3 μl ofcorresponding template. Templates were: nuclease free water for theNTC—Non Template Control —, Roche hgDNA at 11.7 ng/μl (thus, 35 ng or10000 Genome Equivalents (GE) in 3 μl)—wild type—, and HEC-1(A) wcDNAmixed with Roche hgDNA as follows: 1) 0.047 ng/μl of wcDNA HEC-1(A) in11.7 ng/μl of Roche hgDNA, thus 0.14 ng of wcDNA HEC-1(A) and 35 ng ofRoche hgDNA in 3 μl, which corresponds to 40 GE HEC-1(A) (only 20 GE aremutated) and 10000 GE of Roche human genomic DNA (i.e. 1 mt in 500 wt);2) 0.023 ng/μl of wcDNA HEC-1(A) in 11.7 ng/μl of Roche hgDNA, thus 0.07ng of wcDNA HEC-1(A) and 35 ng of Roche hgDNA in 3 μl, which correspondsto 20 GE HEC-1(A) (only 10 GE are mutated) and 10000 GE of Roche hgDNA(i.e. 1 mt in 1000 wt); 3) 0.0117 ng/μl of wcDNA HEC-1(A) in 11.7 ng/μlof Roche hgDNA, thus 0.0035 ng of wcDNA HEC-1(A) and 35 ng of RochehgDNA in 3 μl, which corresponds to 10 GE HEC-1(A) (only 5 GE aremutated) and 10000 GE of Roche hgDNA (i.e. 1 mt in 2000 wt); 4) 0.006ng/μl of wcDNA HEC-1(A) in 11.7 ng/μl of Roche hgDNA, thus 0.00175 ng ofwcDNA HEC-1(A) and 35 ng of Roche hgDNA in 3 μl, which corresponds to 5GE HEC-1(A) (only 2 or 3 GE are mutated) and 10000 GE of Roche hgDNA(i.e. 1 mt in 5000 wt). Note: after preparing the 0.047 ng/μl of wcDNAHEC-1(A) in 11.7 ng/μl of Roche hgDNA mix (40 GE of mutant in 10000 GEof Roche hgDNA), rest of mutant plus Roche hgDNA mixes are prepared byserial dilutions mixing 0.5 volumes of the preceding mutant-Roche hgDNAmix plus 0.5 volumes of Roche hgDNA at 11.7 ng/μl, so the mutant GE arediluted ½ while Roche hgDNA GE remains undiluted (10000 GE). PCRreactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Mass. and thefollowing program: 30 min at 37° C., 2 min at 95° C., 35 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C.

The LDR step was performed in a 10 μl reaction prepared by adding: 5.82μl of nuclease free water (IDT), 1 μl of 10× AK16D ligase reactionbuffer [1× buffer contains 20 mM Tris-HCl pH 8.5 (Bio-Rad, Hercules,Calif.), 5 mM MgCl₂ (Sigma-Aldrich, St. Louis, Mo.), 50 mM KCl(Sigma-Aldrich, St. Louis, Mo.), 10 mM DTT (Sigma-Aldrich, St. Louis,Mo.) and 20 ug/ml of BSA (Sigma-Aldrich, St. Louis, Mo.)], 0.25 μl ofDTT (Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl ofNAD⁺(Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of RNAseH2 (IDT)at 20 mU/μl, 0.2 μl of iCDx-305-P53-248(3)-L Up Rm Up primer at 500 nM,0.2 μl of iCDx-202-P53-248-L Dn P Dn primer at 500 nM, 0.028 μl ofpurified AK16D ligase at 8.8 μM, and 2 μl of PCR reaction. LDR reactionswere run in BioExcell Clear 96 Well PCR 0.2 ml plates (Worldwide MedicalProducts, Inc., Bristol, Pa.) sealed with MicroAmp® clear adhesive filmfrom Applied Biosystems (Applied Biosystems/ThermoFisher; Waltham,Mass.), using a Proflex PCR system thermocycler AppliedBiosystems/ThermoFisher; Waltham, Mass. and the following program: 20cycles of (10 sec at 94° C., and 4 min at 60° C.) followed by a finalhold at 4° C.

The qPCR step was performed in a 10 μl reaction prepared by adding: 1.5μl of nuclease free water (IDT), 5 μl of 2× TaqMan® Fast Universal PCRMaster Mix (fast amplitaq, UDG and dUTP) from Applied Biosystems(Applied Biosystems/ThermoFisher; Waltham, Mass.), 1 μl of iCDx-82GTT-GCGC A2 forward primer at 2.5 μM, 1 μl of iCDx-244-C2 reverse primerat 2.5 μM, 0.5 μl of iCDx-228-p53-248_Probe_s Taqman probe at 5 μM, and1 μl of LDR reaction. qPCR reactions were run in a ViiA7 real-timethermocycler from Applied Biosystems (Applied Biosystems/ThermoFisher;Waltham, Mass.), using MicroAmp® Fast-96-Well Reaction 0.1 ml platessealed with MicroAmp™ Optical adhesive film AppliedBiosystems/ThermoFisher; Waltham, Mass., and the following setting: fastblock, Standard curve as experiment type, ROX as passive reference, Ctas quantification method (automatic threshold, but adjusted to 0.04 whenneeded), FAM as reporter, and NFQ-MGB as quencher; and using thefollowing program: 2 min at 50° C., and 40 cycles of (1 sec at 95° C.,and 20 sec at 60° C.). Results for the experiment using PNA-p53-248-10blocking primer in the PCR step are shown in FIG. 162 and Table 6, andresults for the experiment using PNA-p53-248-11L blocking primer in thePCR step are shown in FIG. 163 and Table 7.

TABLE 6 Results of dilution experiments to detect TP53 R248Q usingPNA-p53-248-10 as blocking primer in the PCR step Starting Genome CtDifference vs Equivalents 10000 GE of Templates (per 10 μl of PCR) CtRoche hgDNA HEC-1(A)_1 (TP53 40 (20 mt) + 6.4 7.3 R248Q, het) + 10,000wt (i.e. 1 mt/ Roche hgDNA (WT) 500 wt) HEC-1(A)_1 (TP53 20 (10 mt) +7.6 6.0 R248Q, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 1,000 wt)HEC-1(A)_1 (TP53 10 (5 mt) + 10.6 3.0 R248Q, het) + 10,000 wt (i.e. 1mt/ Roche hgDNA (WT) 2,000 wt) HEC-1(A)_1 (TP53 5 (2 or 3 mt) + 11.2 2.5R248Q, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 5,000 wt) RochehgDNA (WT) 10,000 wt 13.7 NTC N/A UD Mt = mutant TP53 R248Q. WT =wild-type TP53. Het = heterozygote. UD = Undetermined. Ct values in boldcorrespond to Cts obtained from “true curves”, and values in normal fonttype correspond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

TABLE 7 Results of dilution experiments to detect TP53 R248Q usingPNA-p53-248-11L as blocking primer in the PCR step Starting Genome CtDifference vs Equivalents 10000 GE of Templates (per 10 μl of PCR) CtRoche hgDNA HEC-1(A)_1 (TP53 40 (20 mt) + 6.2 14.5 R248Q, het) + 10,000wt (i.e. 1 mt/ Roche hgDNA (WT) 500 wt) HEC-1(A)_1 (TP53 20 (10 mt) +15.7 5.0 R248Q, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 1,000 wt)HEC-1(A)_1 (TP53 10 (5 mt) + 14.5 6.3 R248Q, het) + 10000 (i.e. 1 mt/Roche hgDNA (WT) 2,000 wt) HEC-1(A)_1 (TP53 5 (2 or 3 mt) + 16.6 4.1R248Q, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 5,000 wt) RochehgDNA (WT) 10,000 wt 20.7 NTC N/A UD Mt = mutant TP53 R248Q. WT =wild-type TP53. Het = heterozygote. UD = Undetermined. Ct values in boldcorrespond to Cts obtained from “true curves”, and values in normal fonttype correspond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

Pixel experiments using hgDNA from Roche. The PCR step was performed ina 130 μl mixture prepared by adding: 56.54 μl of nuclease free water(IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium (Promega,Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison, Wis.), 2.6μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each) (Promega,Madison, Wis.), 3.25 μl of iCDx-326-p53-248 PF WT blk2 forward primer at204, 3.25 μl of iCDx-248-p53-248_PR reverse primer at 204, 16.25 μl ofPNA-p53-248-10 PNA blocking primer at 204, 3.25 μl of RNAseH2 (IDT) at20 mU/μl (diluted in RNAseH2 dilution buffer from IDT), 2.6 μl ofAntarctic thermolabile UDG (New England Biolabs (NEB), Ipswich, Mass.)at 1 U/μl, and 2.86 μl of Klentaql polymerase (DNA PolymeraseTechnology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μ1 to 3 μl of Platinum Taq Antibody at 5U/μl), and 3 μl of corresponding template. Templates were: nuclease freewater for the NTC—Non Template Control—, Roche hgDNA at 2.925 ng/μl(thus, 8.75 ng of Roche hgDNA or 2500 Genome Equivalents (GE) in 3μl)—wild type—, and HEC-1(A) wcDNA mixed with Roche hgDNA as follows: 1)0.023 ng/μl of wcDNA HEC-1(A) in 2.925 ng/μl of Roche hgDNA, thus 0.07ng of wcDNA HEC-1(A) and 8.75 ng of Roche hgDNA in 3 μl, whichcorresponds to 20 GE HEC-1(A) (only 10 GE are mutated) and 2500 GE ofRoche hgDNA; 2) 0.0117 ng/μl of wcDNA HEC-1(A) in 2.925 ng/μl of RochehgDNA, thus 0.0035 ng of wcDNA HEC-1(A) and 8.75 ng of Roche hgDNA in 3μl, which corresponds to 10 GE HEC-1(A) (only 5 GE are mutated) and 2500GE of Roche hgDNA. Note: the 0.0117 ng/μl of wcDNA HEC-1(A) in 2.925ng/μl of Roche hgDNA mix is prepared by serial dilution mixing 0.5volume of the 0.023 ng/μl of wcDNA HEC-1(A) in 2.925 ng/μl of RochehgDNA mix (20 GE of mutant, 10 GE mutated, in 2500 GE of Roche hgDNA)plus 0.5 volume of Roche hgDNA at 2.925 ng/μl, so the mutant is dilutedto 10 GE (5 GE mutated) while Roche hgDNA GE remains undiluted (2500GE).

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Mass. and thefollowing program: 30 min at 37° C., 2 min at 95° C., 35 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section.Results are shown in FIG. 164 and Table 8.

TABLE 8 Results of pixel experiments to detect TP53 R248Q diluted inRoche hgDNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 9 1011 12 Amplifications HEC-1(A)_1 (TP53 20 GE (10 mt) 34.8 18.6 30.0 27.520.3 18.3 18.7 17.4 21.8 17.0 19.5 19.0 9 R248Q, het) + + Roche hgDNA(wt) 2500 GE (wt) HEC-1(A)_1 (TP53 10 GE (5 mt) 19.9 20.2 22.2 UD 19.2UD 18.0 34.7 34.4 19.7 31.3 18.8 7 R248Q, het) + + Roche hgDNA (wt) 2500GE (wt) Roche hgDNA (wt) 2500 GE (wt) 37.0 28.8 33.4 28.8 33.2 UD 32.434.8 UD 34.2 20.5 34.1 1 NTC N/A UD UD UD UD UD UD UD UD UD UD UD UD 0Mt = mutant TP53 R24Q. WT = wild-type TP53. Het = heterozygote. UD =Undetermined. Columns 1-12 correspond to the Cts obtained from tubes1-12, respectively, with values in bold corresponding to Cts obtainedfrom “true curves”, and values in normal font type corresponding tovalues obtained from “flat curves,” i.e. non-baseline curves that appearin the late amplification cycles (generally after a Ct value of 36).

Pixel experiments using human plasma DNA (plasma #10). The PCR step wasperformed in a 130 μl mixture prepared by adding: 46.54 μl of nucleasefree water (IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium(Promega, Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison,Wis.), 2.6 μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each)(Promega, Madison, Wis.), 3.25 μl of iCDx-326-p53-248_PF_WT_blk2 forwardprimer at 2 μM, 3.25 μl of iCDx-248-p53-248_PR reverse primer at 2 μM,16.25 μl of PNA-p53-248-11L PNA blocking primer at 2 μM, 3.25 μl ofRNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT),2.6 μl of Antarctic thermolabile UDG (New England Biolabs (NEB),Ipswich, Mass.) at 1 U/μl, and 2.86 μl of Klentaql polymerase (DNAPolymerase Technology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μl to 3 μl of Platinum Taq Antibody at 5U/μl), and 13 μl of corresponding template. Templates were: nucleasefree water for the NTC—Non Template Control—; Plasma DNA (prepared as7.6 μl nuclease free H2O plus 5.4 μl of plasma DNA at 0.811 ng/μl thus,4.375 ng of plasma DNA or 1250 Genome Equivalents (GE) in the PCRreaction)—wild type—; and HEC-1(A) wcDNA mixed with Plasma DNA asfollows: 1) 5.6 μl nuclease free H2O, plus 5.4 μl Plasma DNA at 0.811ng/μl, plus 2 μl of 0.035 ng/μl of wcDNA HEC-1(A), thus, 4.375 ng ofplasma DNA or 1250 Genome Equivalents (GE) plus 0.07 ng of wcDNAHEC-1(A) which corresponds to 20 GE HEC-1(A) (only 10 GE are mutated) inthe PCR reaction; 2) 6.6 μl nuclease free H2O, plus 5.4 μl Plasma DNA at0.811 ng/μl, plus 1 μl of 0.035 ng/μl of wcDNA HEC-1(A), thus, 4.375 ngof plasma DNA or 1250 Genome Equivalents (GE) plus 0.035 ng of wcDNAHEC-1(A) which corresponds to 10 GE HEC-1(A) (only 5 GE are mutated) inthe PCR reaction. Note: The mix with 4.375 ng of plasma DNA or 1250Genome Equivalents (GE) plus 0.035 ng of wcDNA (10 GE of mutant, 5mutated) is prepared by serial dilution mixing 0.5 volumes of theprevious mix with 4.375 ng of plasma DNA or 1250 Genome Equivalents (GE)and 0.07 ng of wcDNA (20 GE of mutant, 10 mutated) with 0.5 volumes ofthe 4.375 ng of plasma DNA mix.

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler (Applied Biosystems/ThermoFisher; Waltham, Mass.) and thefollowing program: 30 min at 37° C., 2 min at 95° C., 35 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section.Results are shown in FIG. 165 and Table 9.

TABLE 9 Results of pixel experiments to detect TP53 R248Q diluted inhuman plasma DNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 910 11 12 Amplifications HEC-1(A)_1 20 GE (10 mt) 19.7 19.5 22.9 24.020.4 18.7 22.7 23.0 19.3 19.7 20.8 UD 11 (TP53 R248Q, + het) + 1250 GEHuman plasma (wt) DNA (WT) HEC-1(A)_1 10 GE (5 mt) UD UD 19.9 UD 37.1 UD19.7 25.9 22.9 UD 37.4 UD 4 (TP53 R248Q, + het) + 1250 GE Human plasma(wt) UD UD UD UD UD UD UD UD UD UD UD UD 0 DNA (WT) 1250 GE Human plasma(wt) DNA (WT) NTC N/A UD UD UD UD UD UD UD UD UD UD UD UD 0 Mt = mutantTP53 R248Q. WT = wild-type TP53. Het = heterozygote. UD = Undetermined.Columns 1-12 correspond to the Cts obtained from tubes 1-12,respectively, with values in bold corresponding to Cts obtained from“true curves”, and values in normal font type corresponding to valuesobtained from “flat curves,” i.e. non-baseline curves that appear in thelate amplification cycles (generally after a Ct value of 36).

Empirical Example 3 Detection of KRAS Codon 12 First Position Mutations:G12C (34G>T) and G12S (34G>A)

All primers used are listed in Table 10. All primers were purchased fromIntegrated DNA Technologies Inc. ((IDT), Coralville, Iowa), except forthe PNA primer, which was purchased from PNABio Inc. (Thousand Oaks,Calif.).

TABLE 10 Primers for PCR-LDR-qPCR detection of KRASG12C and G12S mutations Primer Name Step Primer Sequence iCDx-327-Kr_12_PCR TGACTGAATATAAACTTGTGGTAGT 2_PF_WT_blk2 TGGArGCTGG/3SpC3/ (SEQ ID NO: 18) iCDx-303-Kr- PCR GGTGTCGTGGCGTCCACAAAATGAT 12_1&2_PRTCTGAATTAGCTGTA  (SEQ ID NO: 19) PNA-Kras 12_2-11L PCRGAGCTGGTGGC (SEQ ID NO: 20) PNA-Kras 12_2-11R PCRAGCTGGTGGCG (SEQ ID NO: 21) iCDx-393-Kr-12_1 LDRTTCGTACCTCGGCACACCAACATAACTG (3)-L_Up_Rm AATATAAACTTGTGGTAGTTGGAGTTHrGTGAT/3SpC3/ (SEQ ID NO: 22) iCDx-222-Kr- LDR/5Phos/GTGGCGTAGGCAAGAGTGCCT 12_1-L_Dn_P TGACGGCGTGTGGCTCCGTTACTCTGTCGA (SEQ ID NO: 23) iCDx-245_A3 qPCR TTCGTACCTCGGCACACCA  (SEQ ID NO: 24)iCD x-246-C3 qPCR TCGACAGAGTAACGGAGCCA  (SEQ ID NO: 25) iCDx-259-T-Kr-qPCR 5′-/5HEX/TT GGA GTT H/ZEN/ 12_1_Probe GT GGC GTA GGC AAG A/3IABk FQ/-3′ (SEQ ID NO: 26) PNA = Peptide nucleic acid, /5SpC3/-5′ C3 Spacer,/3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ Fam FlourescentDye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ Flourescent Quencher ™,/3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green to pink spectralrange, “+”-Locked Nucleic Acid base, “rA”-ribonucleotide baseriboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

Dilution experiments. The PCR step was performed in a 10 μl reactionprepared by adding: 1.58 μl of nuclease free water (IDT), 2 μl of GotaqFlexi buffer 5× without Magnesium (Promega, Madison, Wis.), 0.8 μl ofMgCl₂ at 25 mM (Promega, Madison, Wis.), 0.2 μl of dNTPs (with dATP,dCTP, dGTP and dUTP, 10 mM each) (Promega, Madison, Wis.), 0.25 μl ofiCDx-327-Kr_12_2 PF_WT_blk2 forward primer at 2 μM, 0.25 μl ofiCDx-303-Kr-12_1&2_PR reverse primer at 2 μM, 1.25 μl of PNA-Kras12_2-11L (or PNA-Kras 12_2-11R) PNA blocking primer at 2 μM, 0.25 μl ofRNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT),0.2 μl of Antarctic thermolabile UDG (New England Biolabs (NEB),Ipswich, Mass.) at 1 U/μl, and 0.22 μl of Klentaql polymerase (DNAPolymerase Technology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.02 μl ofKlentaql polymerase at 50 U/μl to 0.2 μl of Platinum Taq Antibody at 5U/μl), and 3 μl of corresponding template. Templates were: nuclease freewater for the NTC—Non Template Control—, Roche hgDNA at 11.7 ng/μl(thus, 35 ng or 10000 Genome Equivalents (GE) in 3 μl)—wild type—, andSW1463 (G12C, 34G>T, homozygotic) or LS123 (G125, 34G>A, heterozygotic)wcDNA mixed with Roche hgDNA as follows: 1) 0.047 ng/μl of SW1463 orLS123 wcDNA in 11.7 ng/μl of Roche hgDNA, thus 0.14 ng of SW1463 orLS123 wcDNA and 35 ng of Roche hgDNA in 3 μl, which corresponds to 40 GEof SW1463 or LS123 wcDNA (40 GE are mutated for SW1463, and 20 GE aremutated for LS123) and 10000 GE of Roche human genomic DNA (i.e. 1 mt in250 wt for SW1463 and 1 mt in 500 wt for LS123); 2) 0.023 ng/μl ofSW1463 or LS123 wcDNA in 11.7 ng/μl of Roche hgDNA, thus 0.07 ng ofSW1463 or LS123 wcDNA and 35 ng of Roche hgDNA in 3 μl, whichcorresponds to 20 GE of SW1463 or LS123 wcDNA (20 GE are mutated forSW1463, and 10 GE are mutated for LS123) and 10000 GE of Roche hgDNA(i.e. 1 mt in 500 wt for SW1463 and 1 mt in 1000 for LS123); 3) 0.0117ng/μl of SW1463 or LS123 wcDNA in 11.7 ng/μl of Roche hgDNA, thus 0.0035ng of SW1463 or LS123 wcDNA and 35 ng of Roche hgDNA in 3 μl, whichcorresponds to 10 GE of SW1463 or LS123 wcDNA (10 GE are mutated forSW1463, and 5 GE are mutated for LS123) and 10000 GE of Roche hgDNA(i.e. 1 mt in 1000 wt for SW1463 and 1 mt in 2000 wt for LS123); 4)0.006 SW1463 or LS123 ng/μl of wcDNA in 11.7 ng/μl of Roche hgDNA, thus0.00175 ng of SW1463 or LS123 wcDNA and 35 ng of Roche hgDNA in 3 μl,which corresponds to 5 GE of SW1463 or LS123 wcDNA (5 GE are mutated forSW1463, and 2 or 3 GE are mutated for LS123) and 10000 GE of Roche hgDNA(i.e. 1 mt in 2000 wt for SW1463 and 1 mt in 5000 wt for LS123). Note:after preparing the 0.047 ng/μl of SW1463 or LS123 wcDNA in 11.7 ng/μlof Roche hgDNA mix (40 GE of mutant in 10000 GE of Roche hgDNA), rest ofmutant plus Roche hgDNA mixes are prepared by serial dilutions mixing0.5 volumes of the preceding mutant-Roche hgDNA mix plus 0.5 volumes ofRoche hgDNA at 11.7 ng/μl so the mutant GE are diluted ½ while RochehgDNA GE remains undiluted (10000 GE). PCR reactions were run inBioExcell Clear 96 Well PCR 0.2 ml plates (Worldwide Medical Products,Inc., Bristol, Pa.) sealed with MicroAmp® clear adhesive film fromApplied Biosystems (Applied Biosystems/ThermoFisher; Waltham, Mass.),using a Proflex PCR system thermocycler Applied Biosystems/ThermoFisher;Waltham, Mass. and the following program: 30 min at 37° C., 2 min at 95°C., 50 cycles of (10 sec at 94° C., 30 sec at 60° C. and 30 sec at 72°C.), 10 min at 99.5° C., and a final hold at 4° C.

The LDR step was performed in a 10 μl reaction prepared by adding: 5.82μl of nuclease free water (IDT), 1 μl of 10× AK16D ligase reactionbuffer [1× buffer contains 20 mM Tris-HCl pH 8.5 (Bio-Rad, Hercules,Calif.), 5 mM MgCl₂ (Sigma-Aldrich, St. Louis, Mo.), 50 mM KCl(Sigma-Aldrich, St. Louis, Mo.), 10 mM DTT (Sigma-Aldrich, St. Louis,Mo.) and 20 ug/ml of BSA (Sigma-Aldrich, St. Louis, Mo.)], 0.25 μl ofDTT (Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl ofNAD⁺(Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of RNAseH2 (IDT)at 20 mU/μl, 0.2 μl of iCDx-393-Kr-12_1(3)-L_Up_Rm Up primer at 500 nM,0.2 μl of iCDx-222-Kr-12_1-L_Dn_P Dn primer at 500 nM, 0.028 μl ofpurified AK16D ligase at 8.8 μM, and 2 μl of PCR reaction. LDR reactionswere run in BioExcell Clear 96 Well PCR 0.2 ml plates (Worldwide MedicalProducts, Inc., Bristol, Pa.) sealed with MicroAmp® clear adhesive filmfrom Applied Biosystems (Applied Biosystems/ThermoFisher; Waltham,Mass.), using a Proflex PCR system thermocycler AppliedBiosystems/ThermoFisher; Waltham, Mass. and the following program: 20cycles of (10 sec at 94° C., and 4 min at 60° C.) followed by a finalhold at 4° C.

The qPCR step was performed in a 10 μl reaction prepared by adding: 1.5μl of nuclease free water (IDT), 5 μl of 2× TaqMan® Fast Universal PCRMaster Mix (fast amplitaq, UDG and dUTP) from Applied Biosystems(Applied Biosystems/ThermoFisher; Waltham, Mass.), 1 μl of iCDx-245_A3forward primer at 2.5 μM, 1 μl of iCDx-246-C3 reverse primer at 2.5 μM,0.5 μl of iCDx-259-T-Kr-12_1 Probe Taqman probe at 504, and 1 μl of LDRreaction. qPCR reactions were run in a ViiA7 real-time thermocycler fromApplied Biosystems (Applied Biosystems/ThermoFisher; Waltham, Mass.),using MicroAmp® Fast-96-Well Reaction 0.1 ml plates sealed withMicroAmp™ Optical adhesive film Applied Biosystems/ThermoFisher;Waltham, Mass., and the following setting: fast block, Standard curve asexperiment type, ROX as passive reference, Ct as quantification method(automatic threshold, but adjusted to 0.04 when needed), HEX asreporter, and NFQ-MGB as quencher; and using the following program: 2min at 50° C., and 40 cycles of (1 sec at 95° C., and 20 sec at 60° C.).Results of the experiment using PNA-Kras 12_2-11L PNA blocking primer inthe PCR step are shown in FIG. 166 and Table 11. Results of theexperiment using PNA-Kras 12_2-11R PNA blocking primer in the PCR stepare shown in FIG. 167 and Table 12.

TABLE 11 Results of dilution experiments to detect KRAS G12C (34G > T)mutation using PNA-Kras 12_2-11L PNA blocking primer in the PCR step.Starting Genome Ct Equivalents Difference vs (per 10 μl of 10000 GE ofTemplates PCR) Ct Roche hgDNA SW1463_1 (KRAS 40 (40 mt) + 26.0 4.9 G12C,hom) + 10,000 wt (i.e. 1 Roche hgDNA (WT) mt/250 wt) SW1463_1 (KRAS 20(20 mt) + 22.1 8.8 G12C, hom) + 10,000 wt (i.e. 1 Roche hgDNA (WT)mt/500 wt) SW1463_1 (KRAS 10 (10 mt) + 24.2 6.7 G12C, hom) + 10,000 wt(i.e. 1 Roche hgDNA (WT) mt/1,000 wt) SW1463_1 (KRAS 5 (5 mt) + 26.3 4.6G12C, hom) + 10,000 wt (i.e. 1 Roche hgDNA (WT) mt/2,000 wt) Roche hgDNA(WT) 10,000 wt 30.9 NTC N/A Undetermined Mt = mutant KRAS G12C. WT =wild-type KRAS. Hom = homozygote. UD = Undetermined. Ct values in boldcorrespond to Cts obtained from “true curves”, and values in normal fonttype correspond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

TABLE 12 Results of dilution experiments to detect KRAS G12S (34G > A)mutation using PNA-Kras 12_2-11R PNA blocking primer in the PCR step.Starting Genome Ct Difference vs Equivalents 10000 GE of Templates (per10 μl of PCR) Ct Roche hgDNA LS123_1 (KRAS 40 (20 mt) + 30.5 1.6 G12S,het) + 10,0000 wt (i.e. 1 mt/ Roche hgDNA (WT) 500 wt) LS123_1 (KRAS 20(10 mt) + 31.2 0.9 G12S, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT)1,000 wt) LS123_1 (KRAS 10 (5 mt) + 31.4 0.7 G12S, het) + 10,000 wt(i.e. 1 mt/ Roche hgDNA (WT) 2,000 wt) LS123_1 (KRAS 5 (2 or 3 mt) +30.5 1.6 G12S, het) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 5,000 wt)Roche hgDNA (WT) 10,000 wt 32.1 NTC N/A 38.4 Mt = mutant KRAS G12S. WT =wild-type KRAS. Het = heterozygote. UD = Undetermined. Ct values in boldcorrespond to Cts obtained from “true curves”, and values in normal fonttype correspond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

Pixel experiments using human plasma DNA (plasma #9). The PCR step wasperformed in a 130 μl mixture prepared by adding: 46.54 μl of nucleasefree water (IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium(Promega, Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison,Wis.), 2.6 μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each)(Promega, Madison, Wis.), 3.25 μl of iCDx-327-Kr 12 2 PF WT blk2 forwardprimer at 2 μM, 3.25 μl of iCDx-303-Kr-12_1&2_PR reverse primer at 2 μM,16.25 μl of PNA-Kras 12_2-11L PNA blocking primer at 2 μM, 3.25 μl ofRNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT),2.6 μl of Antarctic thermolabile UDG (New England Biolabs (NEB),Ipswich, Mass.) at 1 U/μl, and 2.86 μl of Klentaq1 polymerase (DNAPolymerase Technology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μl to 3 μl of Platinum Taq Antibody at 5U/μl), and 13 μl of corresponding template. Templates were: nucleasefree water for the NTC—Non Template Control—; Plasma DNA (prepared as8.3 μl nuclease free H2O plus 4.7 μl of plasma DNA at 0.743 ng/μl thus,3.5 ng of plasma DNA or 1000 Genome Equivalents (GE) in the PCRreaction)—wild type—; and SW1463 wcDNA mixed with Plasma DNA asfollows: 1) 6.3 μl nuclease free H2O, plus 4.7 μl Plasma DNA at 0.743ng/μl, plus 2 μl of 0.0175 ng/μl of wcDNA SW1463, thus, 3.5 ng of plasmaDNA or 1000 Genome Equivalents (GE) plus 0.035 ng of wcDNA SW1463 whichcorresponds to 10 GE SW1463 (all 10 GE are mutated) in the PCR reaction;2) 7.3 μl nuclease free H2O, plus 4.7 μl Plasma DNA at 0.743 ng/μ1, plus1 μl of 0.0175 ng/μl of wcDNA SW1463, thus, 3.5 ng of plasma DNA or 1000Genome Equivalents (GE) plus 0.0175 ng of wcDNA SW1463 which correspondsto 5 GE SW1463 (all 5 GE are mutated) in the PCR reaction. Note: The mixwith 3.5 ng of plasma DNA or 1000 Genome Equivalents (GE) plus 0.0175 ngof wcDNA (5 GE of mutant, 5 mutated) is prepared by serial dilutionmixing 0.5 volumes of the previous mix with 3.5 ng of plasma DNA or 1000Genome Equivalents (GE) and 0.035 ng of wcDNA (10 GE of mutant, 10mutated) with 0.5 volumes of the 3.5 ng of plasma DNA mix.

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Mass. and thefollowing program: 30 min at 37° C., 2 min at 95° C., 50 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section.Results are shown in FIG. 168 and Table 13.

TABLE 13 Results of pixel experiments to detect KRAS G12C diluted inhuman plasma DNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 910 11 12 Amplifications SW1463_1 10 GE (10 mt) UD 22.4 UD UD 18.9 UD38.1 38.8 19.6 37.8 39.6 37.6 3 (KRAS G12C, + hom) + 1000 GE Humanplasma (wt) DNA (WT) SW1463_1 5 GE (5 mt) 38.8 39.7 19.3 25.9 37.2 39.017.9 UD UD 39.6 UD UD 3 (KRAS G12C, + hom) + 1000 GE Human plasma (wt)DNA (WT) Human plasma 1000 GE 39.1 38.4 UD 38.4 39.4 UD 38.3 38.0 UD39.4 UD UD 0 DNA (WT) NTC N/A UD 39.4 UD UD UD 37.8 UD UD UD UD 38.7 UD0 Mt = mutant KRAS G12C. WT = wild-type KRAS. Hom = homozygote UD =Undetermined. Columns 1-12 correspond to the Cts obtained from tubes1-12, respectively, with values in bold corresponding to Cts obtainedfrom “true curves”, and values in normal font type corresponding tovalues obtained from “flat curves,” i.e. non-baseline curves that appearin the late amplification cycles (generally after a Ct value of 36).

Empirical Example 4 Detection of KRAS Codon 12 Second PositionMutations: G12D (35G>A), G12A (35G>C) and G12V (35G>T)

All primers used are listed in Table 14. All primers were purchased fromIntegrated DNA Technologies Inc. ((IDT), Coralville, Iowa), except forthe PNA primer, which was purchased from PNABio Inc. (Thousand Oaks,Calif.).

TABLE 14  Primers for PCR-LDR-qPCR detection of KRASG12D, G12A and G12V mutations Primer Name Step Primer SequenceiCDx-327-Kr_ PCR TGACTGAATATAAACTTGTGGTAGTT 12_2_PF_WT_blk2GGArGCTGG/3SpC3/  (SEQ ID NO: 18) iCDx-303-Kr- PCRGGTGTCGTGGCGTCCACAAAATGATTCT 12_1&2_PR GAATTAGCTGTA (SEQ ID NO: 19)PNA-Kras_12_2-11L PCR GAGCTGGTGGC (SEQ ID NO: 20) iCDx-307-Kr- LDRTTCGTACCTCGGCACACCAACATATG 12_2(3)-L_Up_Rm AATATAAACTTGTGGTAGTTGGAGCCGHrUGGCA/3SpC3/ (SEQ ID NO: 27) iCDx-394-Kr- LDRTTCGTACCTCGGCACACCAACATATG 12_2(3)-L_Up_Rm AATATAAACTTGTGGTAGTTGGAGCCGHrUGGTA/3SpC3/ (SEQ ID NO: 28) iCDx-269-Kr- LDR/5Phos/TGGCGTAGGCAAGAGTGCCTTG 12_2-L_Dn_P ACGGCGTGTGGCTCCGTTACTCTGTCGA (SEQ ID NO: 29) iCDx-245 A3 qPCR TTCGTACCTCGGCACACCA (SEQ ID NO: 24)iCDx-246-C3 qPCR TCGACAGAGTAACGGAGCCA (SEQ ID NO: 25) iCDx-270-Kr-e qPCR5′-/5HEX/TAG TTG GAG/ZEN/CCG 12_2(3)_Prob HTG GCG TAG G/3IABkFQ/-3′(SEQ ID NO: 30) PNA = Peptide nucleic acid, /5SpC3/-5′ C3 Spacer,/3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ Fam FlourescentDye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ Flourescent Quencher ™,/3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green to pink spectralrange, “+”-Locked Nucleic Acid base, “rA”-ribonucleotide baseriboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

Dilution experiments. The PCR step was performed in a 10 μl reactionprepared by adding: 1.58 μl of nuclease free water (IDT), 2 μl of GotaqFlexi buffer 5× without Magnesium (Promega, Madison, Wis.), 0.8 μl ofMgCl₂ at 25 mM (Promega, Madison, Wis.), 0.2 μl of dNTPs (with dATP,dCTP, dGTP and dUTP, 10 mM each) (Promega, Madison, Wis.), 0.25 μl ofiCDx-327-Kr_12_2_PF_WT_blk2 forward primer at 2 μM, 0.25 μl ofiCDx-303-Kr-12_1&2_PR reverse primer at 2 μM, 1.25 μl of PNA-Kras12_2-11L PNA blocking primer at 2 μM, 0.25 μl of RNAseH2 (IDT) at 20mU/μl (diluted in RNAseH2 dilution buffer from IDT), 0.2 μl of Antarcticthermolabile UDG (New England Biolabs (NEB), Ipswich, Mass.) at 1 U/μl,and 0.22 μl of Klentaql polymerase (DNA Polymerase Technology, St.Louis, Mo.) mixed with Platinum Taq Antibody (Invitrogen, Carlsbad,Calif.) (the mix is prepared by adding 0.02 μl of Klentaq1 polymerase at50 U/μl to 0.2 μl of Platinum Taq Antibody at 5 U/μl), and 3 μl ofcorresponding template. Templates were: nuclease free water for theNTC—Non Template Control—, Roche hgDNA at 11.7 ng/μl (thus, 35 ng or10000 Genome Equivalents (GE) in 3 μl)—wild type—, HEC-1(A) (G12D,35G>A, heterozygotic) or SW1116 (G12A, 35G>C, heterozygotic) or SW480(G12V, 35G>T, homozygotic) wcDNA mixed with Roche hgDNA as follows: 1)0.047 ng/μl of HEC-1(A), SW1116 or SW480 wcDNA in 11.7 ng/μl of RochehgDNA, thus 0.14 ng of HEC-1(A), SW1116 or SW480 and 35 ng of RochehgDNA in 3 μl, which corresponds to 40 GE of HEC-1(A), SW1116 or SW480(20 GE are mutated for HEC-1(A) or SW1116, and 40 GE are mutated forSW480) and 10000 GE of Roche human genomic DNA (i.e. 1 mt in 500 wt forHEC-1(A) or SW1116 and 1 mt in 250 for SW480; 2) 0.023 ng/μl of wcDNA ofHEC-1(A), SW1116 or SW480 in 11.7 ng/μl of Roche hgDNA, thus 0.07 ng ofHEC-1(A), SW1116 or SW480 wcDNA and 35 ng of Roche hgDNA in 3 μl, whichcorresponds to 20 GE of HEC-1(A), SW1116 or SW480 (10 GE are mutated forHEC-1(A) or SW1116, and 20 GE are mutated for SW480) and 10000 GE ofRoche hgDNA (i.e. 1 mt in 1000 wt for HEC-1(A) or SW1116 and 1 mt in 500wt for SW480); 3) 0.0117 ng/μl of HEC-1(A), SW1116 or SW480 wcDNA in11.7 ng/μl of Roche hgDNA, thus 0.0035 ng of HEC-1(A), SW1116 or SW480wcDNA and 35 ng of Roche hgDNA in 3 μl, which corresponds to 10 GE ofHEC-1(A), SW1116 or SW480 (5 GE are mutated for HEC-1(A) or SW1116, and10 GE are mutated for SW480) and 10000 GE of Roche hgDNA (i.e. 1 mt in2000 for HEC-1(A) or SW1116 and 1 mt in 1000 for SW480); 4) 0.006 ng/μlof HEC-1(A), SW1116 or SW480 wcDNA in 11.7 ng/μl of Roche hgDNA, thus0.00175 ng of HEC-1(A), SW1116 or SW480 wcDNA and 35 ng of Roche hgDNAin 3 μl, which corresponds to 5 GE of HEC-1(A), SW1116 or SW480 (2 or 3GE are mutated for HEC-1(A) or SW1116, and 5 GE are mutated for SW480)and 10000 GE of Roche hgDNA (i.e. 1 mt in 5000 wt for HEC-1(A) or SW1116and 1 mt in 2000 wt for SW480). Note: after preparing the 0.047 ng/μl ofHEC-1(A), SW1116 or SW480 wcDNA in 11.7 ng/μl of Roche hgDNA mix (40 GEof mutant in 10000 GE of Roche hgDNA), rest of mutant plus Roche hgDNAmixes are prepared by serial dilutions mixing 0.5 volumes of thepreceding mutant-Roche hgDNA mix plus 0.5 volumes of Roche hgDNA at 11.7ng/μl so the mutant GE are diluted ½ while Roche hgDNA GE remainsundiluted (10000 GE). PCR reactions were run in BioExcell Clear 96 WellPCR 0.2 ml plates (Worldwide Medical Products, Inc., Bristol, Pa.)sealed with MicroAmp® clear adhesive film from Applied Biosystems(Applied Biosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCRsystem thermocycler (Applied Biosystems/ThermoFisher; Waltham, Mass.)and the following program: 30 min at 37° C., 2 min at 95° C., 50 cyclesof (10 sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at99.5° C., and a final hold at 4° C.

The LDR step was performed in a 10 μl reaction prepared by adding: 5.82μl of nuclease free water (IDT), 1 μl of 10× AK16D ligase reactionbuffer [1× buffer contains 20 mM Tris-HCl pH 8.5 (Bio-Rad, Hercules,Calif.), 5 mM MgCl₂ (Sigma-Aldrich, St. Louis, Mo.), 50 mM KCl(Sigma-Aldrich, St. Louis, Mo.), 10 mM DTT (Sigma-Aldrich, St. Louis,Mo.) and 20 ug/ml of BSA (Sigma-Aldrich, St. Louis, Mo.)], 0.25 μl ofDTT (Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of NAD⁺(Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of RNAseH2 (IDT) at 20mU/μl, 0.2 μl of iCDx-307-Kr-12_2(3)-L_Up_Rm oriCDx-394-Kr-12_2(3)-L_Up_Rm Up primer at 500 nM, 0.2 μl ofiCDx-269-Kr-12_2-L_Dn_P Dn primer at 500 nM, 0.028 μl of purified AK16Dligase at 8.8 μM, and 2 μl of PCR reaction. LDR reactions were run inBioExcell Clear 96 Well PCR 0.2 ml plates (Worldwide Medical Products,Inc., Bristol, Pa.) sealed with MicroAmp® clear adhesive film fromApplied Biosystems (Applied Biosystems/ThermoFisher; Waltham, Mass.),using a Proflex PCR system thermocycler Applied Biosystems/ThermoFisher;Waltham, Ma and the following program: 20 cycles of (10 sec at 94° C.,and 4 min at 60° C.) followed by a final hold at 4° C.

The qPCR step was performed in a 10 μl reaction prepared by adding: 1.5μl of nuclease free water (IDT), 5 μl of 2× TaqMan® Fast Universal PCRMaster Mix (fast amplitaq, UDG and dUTP) from Applied Biosystems(Applied Biosystems/ThermoFisher; Waltham, Mass.), 1 μl of iCDx-245 A3forward primer at 2.5 μM, 1 μl of iCDx-246-C3 reverse primer at 2.5 μM,0.5 μl of iCDx-270-Kr-12_2(3) Probe Taqman probe at 5 μM, and 1 μl ofLDR reaction. qPCR reactions were run in a ViiA7 real-time thermocyclerfrom Applied Biosystems (Applied Biosystems/ThermoFisher; Waltham,Mass.), using MicroAmp® Fast-96-Well Reaction 0.1 ml plates sealed withMicroAmp™ Optical adhesive film Applied Biosystems/ThermoFisher;Waltham, Mass., and the following setting: fast block, Standard curve asexperiment type, ROX as passive reference, Ct as quantification method(automatic threshold, but adjusted to 0.04 when needed), HEX asreporter, and NFQ-MGB as quencher; and using the following program: 2min at 50oC, and 40 cycles of (1 sec at 95° C., and 20 sec at 60° C.).Results of the experiment to detect KRAS G12D (35G>A) mutation usingiCDx-307-Kr-12_2(3)-L up Rm UP LDR primer are shown in FIG. 169 andTable 15. Results of the experiment to detect KRAS G12A (G>C) mutationusing iCDx-307-Kr-12_2(3)-L up Rm UP LDR primer are shown in FIG. 170and Table 16. Results of the experiment to detect KRAS G12V (35G>T)mutation using iCDx-307-Kr-12_2(3)-L up Rm UP LDR primer are shown inFIG. 171 and Table 17.

TABLE 15 Results of dilution experiments to detect KRAS G12D (35G > A)mutation using iCDx-307-Kr-12_2(3)-L_Up_Rm UP LDR primer. StartingGenome Equivalents Ct Difference vs (per 10 μl of 10000 GE of TemplatesPCR) Ct Roche hgDNA HEC-1(A)_1 (KRAS 40 (20 mt) + 28.4 6.0 G12D, het) +10,000 wt (i.e. 1 Roche hgDNA mt/500 wt) HEC-1(A)_1 (KRAS 20 (10 mt) +31.0 3.5 G12D, het) + 10,000 wt (i.e. 1 Roche hgDNA mt/1,000 wt)HEC-1(A)_1 (KRAS 10 (5 mt) + 32.2 2.2 G12D, het) + 10,000 wt (i.e. 1Roche hgDNA mt/2,000 wt) HEC-1(A)_1 (KRAS 5 (2 or 3 mt) + 29.8 4.6 G12D,het) + 10,000 wt (i.e. 1 Roche hgDNA (WT) mt/5,000 wt) Roche hgDNA (WT)10,000 wt 34.4 NTC N/A 35.1 Mt = mutant KRAS G12D. WT = wild-type KRAS.Het = heterozygote. UD = Undetermined. Ct values in bold correspond toCts obtained from “true curves”, and values in normal font typecorrespond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

TABLE 16 Results of dilution experiments to detect KRAS G12A (35G > C)mutation using iCDx-307-Kr-12_2(3)-L_Up_Rm UP LDR primer. StartingGenome Equivalents Ct Difference vs (per 10 μl of 10000 GE of TemplatesPCR) Ct Roche hgDNA SW1116 (KRAS 40 (20 mt) + 24.8 9.6 G12A, het) +10,000 wt (i.e. 1 Roche hgDNA (WT) mt/500 wt) SW1116 (KRAS 20 (10 mt) +23.6 10.8 G12A, het) + 10,000 wt (i.e. 1 Roche hgDNA (WT) mt/1,000 wt)SW1116 (KRAS 10 (5 mt) + 25.2 9.2 G12A, het) + 10,000 wt (i.e. 1 RochehgDNA (WT) mt/2,000 wt) SW1116 (KRAS 5 (2 or 3 mt) + 28.9 5.6 G12A,het) + 10,000 wt (i.e. 1 Roche hgDNA (WT) mt/5,000 wt) Roche hgDNA (WT)10,000 wt 34.4 NTC N/A 36.0 Mt = mutant KRAS G12A. WT = wild-type KRAS.Het = heterozygote. UD = Undetermined. Ct values in bold correspond toCts obtained from “true curves”, and values in normal font typecorrespond to values obtained from “flat curves,” i.e. non-baselinecurves that appear in the late amplification cycles (generally after aCt value of 36).

TABLE 17 Results of dilution experiments to detect KRAS G12V (35G > T)mutation using iCDx-307-Kr-12_2(3)-L_Up_Rm UP LDR primer. StartingGenome Ct Difference vs Equivalents 10000 GE of Templates (per 10 μl ofPCR) Ct Roche hgDNA SW480 (KRAS 40 (40 mt) + 21.9 12.6 G12V, hom) +10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 250 wt) SW480 (KRAS 20 (20 mt) +22.8 11.6 G12V, hom) + 10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 500 wt)SW480 (KRAS 10 (10 mt) + 24.0 10.5 G12V, hom) + 10,000 wt (i.e. 1 mt/Roche hgDNA (WT) 1,000 wt) SW480 (KRAS 5 (5 mt) + 27.9 6.5 G12V, hom) +10,000 wt (i.e. 1 mt/ Roche hgDNA (WT) 2,000 wt) Roche hgDNA (WT) 10,000wt 34.4 NTC N/A 34.8 Mt = mutant KRAS G12V. WT = wild-type KRAS. Hom =homozygote. UD = Undetermined. Ct values in bold correspond to Ctsobtained from “true curves”, and values in normal font type correspondto values obtained from “flat curves,” i.e. non-baseline curves thatappear in the late amplification cycles (generally after a Ct value of36).

Pixel experiments using human plasma DNA (plasma #9). The PCR step wasperformed in a 130 μl mixture prepared by adding: 46.54 μl of nucleasefree water (IDT), 26 μl of Gotaq Flexi buffer 5× without Magnesium(Promega, Madison, Wis.), 10.4 μl of MgCl₂ at 25 mM (Promega, Madison,Wis.), 2.6 μl of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each)(Promega, Madison, Wis.), 3.25 μl of iCDx-327-Kr_12_2_PF_WT_blk2 forwardprimer at 2 μM, 3.25 μl of iCDx-303-Kr-12_1&2_PR reverse primer at 2 μM,16.25 μl of PNA-Kras 12_2-11L PNA blocking primer at 2 μM, 3.25 μl ofRNAseH2 (IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT),2.6 μl of Antarctic thermolabile UDG (New England Biolabs (NEB),Ipswich, Mass.) at 1 U/μl, and 2.86 μl of Klentaql polymerase (DNAPolymerase Technology, St. Louis, Mo.) mixed with Platinum Taq Antibody(Invitrogen, Carlsbad, Calif.) (the mix is prepared by adding 0.3 μl ofKlentaql polymerase at 50 U/μl to 3 μl of Platinum Taq Antibody at 5U/μl), and 13 μl of corresponding template. Templates were: nucleasefree water for the NTC—Non Template Control —; Plasma DNA (prepared as8.3 μl nuclease free H2O plus 4.7 μl of plasma DNA at 0.743 ng/μl thus,3.5 ng of plasma DNA or 1000 Genome Equivalents (GE) in the PCRreaction)—wild type—; and SW480 wcDNA mixed with Plasma DNA asfollows: 1) 6.3 μl nuclease free H2O, plus 4.7 μl Plasma DNA at 0.743ng/μ1, plus 2 μl of 0.0175 ng/μl of SW480 wcDNA, thus, 3.5 ng of plasmaDNA or 1000 Genome Equivalents (GE) plus 0.035 ng of SW480 wcDNA whichcorresponds to 10 GE of SW480 wcDNA (all 10 GE are mutated) in the PCRreaction; 2) 7.3 μl nuclease free H2O, plus 4.7 μl Plasma DNA at 0.743ng/μl, plus 1 μl of 0.0175 ng/μl of SW480 wcDNA, thus, 3.5 ng of plasmaDNA or 1000 Genome Equivalents (GE) plus 0.0175 ng of SW480 wcDNA whichcorresponds to 5 GE of SW480 (all 5 GE are mutated) in the PCR reaction.Note: The mix with 3.5 ng of plasma DNA or 1000 Genome Equivalents (GE)plus 0.0175 ng of SW480 wcDNA (5 GE of mutant, 5 mutated) is prepared byserial dilution mixing 0.5 volumes of the previous mix with 3.5 ng ofplasma DNA or 1000 Genome Equivalents (GE) and 0.035 ng of SW480 wcDNA(10 GE of mutant, 10 mutated) with 0.5 volumes of the 3.5 ng of plasmaDNA mix.

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in BioExcell Clear 96 Well PCR 0.2 ml plates(Worldwide Medical Products, Inc., Bristol, Pa.) sealed with MicroAmp®clear adhesive film from Applied Biosystems (AppliedBiosystems/ThermoFisher; Waltham, Mass.), using a Proflex PCR systemthermocycler Applied Biosystems/ThermoFisher; Waltham, Ma and thefollowing program: 30 min at 37° C., 2 min at 95° C., 50 cycles of (10sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at 99.5°C., and a final hold at 4° C. The subsequent LDR and qPCR step wereperformed as described above, in the Dilution experiments section, usingiCDx-394-Kr-12_2(3)-L_Up_Rm as Upstream primer for the LDR step. Resultsare shown in FIG. 172 and Table 18.

TABLE 18 Results of pixel experiments to detect KRAS G12V diluted inhuman plasma DNA. GE per Total Templates 130 μl of PCR 1 2 3 4 5 6 7 8 910 11 12 Amplifications SW480_1 10 GE (10 mt) 35.5 19.4 21.5 35.6 18.819.3 19.4 34.9 22.6 22.0 20.0 20.5 9 (KRAS G12V, + hom) + 1000 GE Humanplasma (wt) DNA (WT) SW480_1 5 GE (5 mt) 22.4 21.0 20.3 22.3 20.2 35.335.0 19.6 19.8 26.5 35.7 20.4 9 (KRAS G12V, + hom) + 1000 GE Humanplasma (wt) DNA (WT) Human plasma 1000 GE 37.9 36.1 36.3 36.2 35.7 36.236.4 35.5 36.0 37.1 36.3 35.9 0 DNA (WT) (wt) NTC N/A 36.0 36.6 35.936.9 36.2 36.2 36.7 36.5 37.2 36.5 36.8 35.9 0 Mt = mutant KRAS G12V. WT= wild-type KRAS. Hom = homozygote. UD = Undetermined. Columns 1-12correspond to the Cts obtained from tubes 1-12, respectively, withvalues in bold corresponding to Cts obtained from “true curves”, andvalues in normal font type corresponding to values obtained from “flatcurves,” i.e. non-baseline curves that appear in the late amplificationcycles (generally after a Ct value of 36).

Empirical Example 5 Detection of Methylation in Cell Line Genomic DNA

General Methods. The cell lines used were: LS-174T, HCT-15, HT-29, WiDi,SW1116, colon adenocarcinoma cell line. All cell lines were seeded in 60cm² cμlture dishes in McCoy's 5a medium containing 4.5 g/l glucose,supplemented with 10% fetal calf serum and kept in a humidifiedatmosphere containing 5% CO₂. Once cells reached 80-90% confluence theywere washed in Phosphate Buffered Saline (×3) and collected bycentrifugation (500×g). DNA was isolated using the DNeasy Blood & TissueKit (Qiagen; Valencia, Calif.). DNA concentration was determined withQuant-iT Picogreen Assay (Life Technologies/ThermoFisher; Waltham,Mass.).

Human Genomic DNA (0.2 mg/ml) containing high molecμlar weight (>50 kb)genomic DNA isolated from human blood (buffy coat) (Roche human genomicDNA) was purchased from Roche (Indianapolis, Ind.). Its concentrationwas determined to be 39 ng/μl by Quant-iT PicoGreen dsDNA Assay Kit.

Restriction Digestion of Genomic DNA with Enzyme Bsh1236I. Genomic DNA(500 ng) from the cell lines listed above were digested with 10 units ofthe restriction enzyme Bsh1236I in 20 μl of reaction mixture containing1×CutSmart buffer (50 mM Potassium Acetate, 20 mM Tris-Acetate, 10 mMMagnesium Acetate, 100 μg/ml BSA, pH7.9 @ 25° C.). The digestionreactions were carried out at 37° C. for 1 hour and subsequent enzymeinactivation by heating to 80° C. for 20 min.

Bisulfite conversion of digested genomic DNA. Bisulfite conversion wascarried out using the EZ DNA Methylation-Lightning kit from ZymoResearch Corporation (Irvine, Calif.). 130 μl of Lightning ConversionReagent was added to 20 μl of the Bsh1236I digested genomic DNA. Theconversion reaction was incubated at 98° C. for 8 minutes, followed by54° C. for one hour and then cooled down to 4° C. 600 μl of M-BindingBuffer was added to a Zymo-Spin™ Column followed by column placementinto a collection tube. The 150 μl reaction mixture containing digestedDNA and lightning conversion reagent was loaded into the Zymo-Spin ICColumn containing the 600 μl of M-Binding Buffer. The cap of the columnwas sealed, and the solution was mixed by inverting the column severaltimes. The column was centrifuged at full speed (≥10,000×g) for 30 secwith the flow through discarded. 100 μl of M-washing buffer was added tothe column and the column was centrifuged at full speed for 30 secondsand the flow through was discarded. 200 μl of L-Desulphonation Bufferwas added into the column, and the column was allowed to stand at roomtemperature for 15-20 minutes. After the incubation, the column wascentrifuged at full speed for 30 seconds. 200 μl of M-Wash Buffer wasadded to the column and the column was centrifuged at full speed for 30seconds with the flow through discarded. This wash step was repeated onemore time. Finally, the column was placed into a 1.5 ml micro centrifugetube, and 10 μl of M-Elution buffer was added to the column matrix andcentrifuged at full speed for 30 second to elute the Bisulfite convertedDNA.

PCR and LDR primers. All primers used are listed in Table 19. Allprimers were purchased from Integrated DNA Technologies Inc. ((IDT),Coralville, Iowa), except for LNA1 and LNA2, which was purchased fromExiqon Inc. (Woburn, Mass.), and PNA, which was purchased from PNA Bio(Thousand Oaks, Calif.).

TABLE 19 Primers for PCR-LDR-qPCR methylation. Primer Name StepPrimer Sequence iCDx-2031- PCR GAACTCCAACCGAAACTACGTA Vim-S3-FPArCTACA/3SpC3/  (SEQ ID NO: 31) iCDx-2032A- PCR GGTGTCGTGGACGAGGCGTAGAVim-S3-RP GGTTGCrGGTTA/3SpC/  (SEQ ID NO: 32) VIM-53-LNA1 PCRCA+TAA+CT+AC+AT+CC+AC+ CCA (SEQ ID NO: 33) VIM-S3-LNA2 PCRCA+TAA+CT+AC+AT+C+C+AC+ CCA (SEQ ID NO: 34) VIM-S3-PNA2 PCRACATAACTACATCCACCCA  (SEQ ID NO: 35) iCDx-2033A- LDRTAGACACGAGCGAGGTCACAACT Vim-53-Up CCAACCGAAACTACGTAACTGCG rUCCGT/3SpC3/ (SEQ ID NO: 36) iCDx-2034A- LDR /5Phos/TCCACCCGCACCTACA Vim-53-DnACCTAAACAACGCGTGCAAAATT CAGGCTGTGCA  (SEQ ID NO: 37) iCDx-2035A- qPCRTAACTGCGTCCACCCGCACCTAC Vim-53-RT-Pb (SEQ ID NO: 38) iCDx-2036- qPCRTAGACACGAGCGAGGTCAC  Vim-53-RT-FP (SEQ ID NO: 39) iCDx-2037- qPCRTGCACAGCCTGAATTTTGCAC  Vim-53-RT-RP (SEQ ID NO: 40) /5SpC3/-5′ C3Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ FamFlourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ FlourescentQuencher ™, /3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green topink spectral range, “+”-Locked Nucleic Acid base, “rA”-ribonucleotidebase riboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

PCR-LDR-qPCR experiments. The PCR step was performed in a 10 μl reactionprepared by adding: 2 μl of Gotaq Flexi buffer 5× without Magnesium(Promega, Madison, Wis.), 0.8 μl of MgCl₂ at 25 mM (Promega, Madison,Wis.), 0.41 of dNTPs (with dATP, dCTP, dGTP and dUTP, 10 mM each)(Promega, Madison, Wis.), 0.25 μl of iCDx-2031-Vim-S3-FP forward primerat 2 μM, 0.25 μl of iCDx-2032A-Vim-S3-RP reverse primer at 2 μM, 1.25 μlof VIM-S3-LNA or VIM-S3-PNA2 blocking primer at 2 μM, 0.25 μl of RNAseH2(IDT) at 20 mU/μl (diluted in RNAseH2 dilution buffer from IDT), and0.22 μl of Klentaql polymerase (DNA Polymerase Technology, St. Louis,Mo.) mixed with Platinum Taq Antibody (Invitrogen/Thermo Fisher,Waltham, Mass.) (the mixture is prepared by adding 0.02 μl of Klentaqlpolymerase at 50 U/μl to 0.2 μl of Platinum Taq Antibody at 5 U/μl), and4.78 μl of corresponding template containing 35 ng genomic DNA.Templates were: LS-174T, HCT-15, HT-29, WiDr, SW1116 cell line genomicDNA, and Roche human genomic DNA. PCR reactions were run in a ProFlexPCR system thermocycler (Applied Biosystems/ThermoFisher, Waltham,Mass.) and run with the following program: 2 min at 95° C., 40 cycles of(10 sec at 94° C., 30 sec at 60° C. and 30 sec at 72° C.), 10 min at99.5° C., and a final hold at 4° C.

The LDR step was performed in a 10 μl reaction prepared by adding: 5.82μl of nuclease free water (IDT), 1 μl of 10× AK16D ligase reactionbuffer [1× buffer contains 20 mM Tris-HCl pH 8.5 (Bio-Rad, Hercules,Calif.), 5 mM MgCl₂ (Sigma-Aldrich, St. Louis, Mo.), 50 mM KCl(Sigma-Aldrich, St. Louis, Mo.), 10 mM DTT (Sigma-Aldrich, St. Louis,Mo.) and 20 ug/ml of BSA (Sigma-Aldrich, St. Louis, Mo.)], 0.25 μl ofDTT (Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of NAD⁺(Sigma-Aldrich, St. Louis, Mo.) at 40 mM, 0.25 μl of RNAseH2 (IDT) at 20mU/μl, 0.2 μl of iCDx-2033A-Vim-S3-Up primer at 500 nM, 0.2 μl ofiCDx-2034A-Vim-S3-Dn primer at 500 nM, 0.028 μl of purified AK16D ligaseat 8.8 μM, and 2 μl of PCR reaction. LDR reactions were run in a ProFlexPCR system thermocycler (Applied Biosystems/ThermoFisher; Waltham,Mass.) and the following program: 20 cycles of (10 sec at 94° C., and 4min at 60° C.) followed by a final hold at 4° C.

The qPCR step was performed in a 10 μl reaction mixture prepared byadding: 1.5 μl of nuclease free water (IDT), 5 μl of 2× TaqMan® FastUniversal PCR Master Mix (Fast amplitaq, UDG and dUTP) from AppliedBiosystems (Applied Biosystems/ThermoFisher; Waltham, Mass.), 1 μl ofiCDx-2036-Vim-S3-RT-FP forward primer at 2.5 μM, 1 μl ofiCDx-2037-Vim-S3-RT-RP reverse primer at 2.5 μM, 0.5 μl ofiCDx-2035A-Vim-S3-RT-Pb Taqman probe at 5 μM, and 1 μl of LDR reactionproducts. qPCR reactions were run in a ViiA7 real-time thermo-cyclerfrom Applied Biosystems (Applied Biosystems/ThermoFisher; Waltham,Mass.), using MicroAmp® Fast-96-Well Reaction 0.1 ml plates sealed withMicroAmp™ Optical adhesive film (Applied Biosystems/ThermoFisher;Waltham, Mass.), and the following setting: fast block, Standard curveas experiment type, ROX as passive reference, Ct as quantificationmethod (automatic threshold, but adjusted to 0.05 when needed), TAMRA asreporter, and NFQ-MGB as quencher; and using the following program: 2min at 50° C., and 40 cycles of (1 sec at 95° C., and 20 sec at 60° C.).Results are shown in FIG. 173 and Table 20.

TABLE 20 Results of experiments to detect methylation in cell linegenomic DNA. Starting Genome Ct Difference vs Equivalents 10000 GE ofTemplates (per 10 μl of PCR) Ct Roche hgDNA LS-174T 10,000 28.5 10.1HCT-15 10,000 20.2 18.6 HT-29 10,000 20.4 18.2 WiDr 10,000 20.3 18.3SW1116 10,000 UD 0 Roche DNA 10,000 38.6 0

Pixel experiments. The PCR step was setup in a 130 μl mixture preparedby adding: 59.14 μl of nuclease free water (IDT), 26 μl of Gotaq Flexibuffer 5× without Magnesium (Promega, Madison, Wis.), 10.4 μl of MgCl₂at 25 mM (Promega, Madison, Wis.), 2.6 μl of dNTPs (with dATP, dCTP,dGTP and dUTP, 10 mM each) (Promega, Madison, Wis.), 3.25 μl ofiCDx-2031-VIM-S3-FP forward primer at 2 μM, 3.25 μl ofiCDx-2032-VIM-S3-PR reverse primer at 2 μM, 16.25 μl of iCDx-VIM-S3-LNA2blocking primer at 2 μM, 3.25 μl of RNAseH2 (IDT) at 20 mU/μl (dilutedin RNAseH2 dilution buffer from IDT), 2.6 μl of Antarctic thermolabileUDG (New England Biolabs (NEB), Ipswich, Mass.) at 1 U/μl, and 2.86 μlof Klentaql polymerase (DNA Polymerase Technology, St. Louis, Mo.) mixedwith Platinum Taq Antibody (Invitrogen, Carlsbad, Calif.) (the mixtureis prepared by adding 0.3 μl of Klentaq1 polymerase at 50 U/μl to 3 μlof Platinum Taq Antibody at 5 U/μl), and 3 μl of corresponding template.3 μl of templates contains: (1) 0.070 ng (20 GE) HT-29 DNA mixed with 9ng (2500 GE) Roche hgDNA. (2) 0.035 ng (10 GE) HT-29 cell line DNA mixedwith 9 ng (2500 GE) Roche hgDNA. (3) 9 ng (2500 GE) Roche DNA, (4)nuclease free water for the Non Template Control (NTC).

Each 130 μl PCR mixture was divided into 12 tubes, 10 μl each, and thenthe PCR reactions were run in a Proflex PCR system thermo-cycler(Applied Biosystems/ThermoFisher; Waltham, Mass.) and the followingprogram: 2 min at 95° C., 40 cycles of (10 sec at 94° C., 30 sec at 60°C. and 30 sec at 72° C.), 10 min at 99.5° C., and a final hold at 4° C.The subsequent LDR and qPCR step were performed as described above.Results are shown in FIG. 174 and Table 25.

The PCR-LDR-qPCR procedure for methylation detection in VIM-S3 top andbottom strands was repeated as described above using a modified versionof the LDR and Taqman probes (i.e., version B) probes. A comparison ofresults for methylation detection is shown in the amplification plots ofFIGS. 175-178. FIG. 175 provides the real-time PCR plots for cell-lineand wild-type (Roche) DNA using the VIM-S3 top-strand primer design andthe Taqman probe version “A” (Table 21). Results show a Ct value ofabout 10 and 11.5 for WiDr and HT-29 cell line DNA respectively,indicating methylation at the S3 site in the VIM promoter region. Celllines SW1116, Roche DNA, no-template control (NTC) for the initial PCRstep, the following LDR step, and the final Taqman step are allbase-line (i.e. flat). The same experiment was repeated using version“A” probes designed for the bottom strand (FIG. 176 and Table 22). Asexpected, results show a Ct value of about 11 and 9 for WiDr and HT-29cell line DNA respectively, indicating methylation at the S3 site in theVIM promoter region. However, results with SW1116, Roche DNA,no-template control (NTC) for the initial PCR step, and NTC for thefollowing LDR step al give Ct values of around 28.5 to 31, even thoughwe know from the prior experiment that there is no methylation at the S3site in the VIM promoter region. This discrepancy was resolved bydesigning the Taqman probes to overlap the upstream LDR probe portion ofthe LDR product by about 9 bases, and the downstream LDR probe portionof the LDR product by the remaining bases in the probe. Further, twonon-complementary bases on the 5′ side were added to the Taqman probe toassure that any accidental cross-polymerase extension would not be ableto extend on the upstream LDR probe in a subsequent round. FIG. 177provides the real-time PCR plots for cell-line and wild-type (Roche) DNAusing the WIM-S3 top-strand primer design and the Taqman probe version“B” (Table 23). Results show a Ct value of about 12 and 13.5 for WiDrand HT-29 cell line DNA respectively, indicating methylation at the S3site in the VIM promoter region. Cell lines SW1116, Roche DNA,no-template control (NTC) for the initial PCR step, the following LDRstep, and the final Taqman step are all base-line (i.e. flat). The sameexperiment was repeated using version “B” probes designed for the bottomstrand (FIG. 178 and Table 24). As expected, results show a Ct value ofabout 11.5 and 9.5 for WiDr and HT-29 cell line DNA respectively,indicating methylation at the S3 site in the VIM promoter region. Now,results with cell lines SW1116, Roche DNA, no-template control (NTC) forthe initial PCR step, the following LDR step, and the final Taqman stepare all base-line (i.e. flat), indicating the new probe design version“B” solved the problem of false signal from NTC or samples withoutmethylation in the VIM-site 3 region.

TABLE 21 Methylation Primers for VIM_S3_Top Strand usingTaqman Probe version “A” Primer Primer  Type Name Sequences PCRiCDx_2031 GAACTCCAACCGAAACTACGTAArCTACA/ 3SpC3/ (SEQ ID NO: 31) PCRiCDx_2032 GGTGTCGTGGACGAGGCGTAGAGGTTGCr GGTTA/3SpC3/ (SEQ ID NO: 32) LDRiCDx_2033A AAACTACGTATAGACACGAGCGAGGTCACA ACTCCAACCGACTGCGrUCCGT/3SpC3/ (SEQ ID NO: 36) LDR iCDx_2034 /5Phos/TCCACCCGCACCTACAACCTAAACAACGCGTGCAAAATTCAGGCTGTGCA  (SEQ ID NO: 37) Taqman iCDx_2035/5HEX/TAACTGCGT/ZEN/CCACCCGCAC Probe CTAC/3IABkFQ/ (SEQ ID NO: 38)Taqman iCDx_2036 TAGACACGAGCGAGGTCAC Primer (SEQ ID NO: 39) TaqmaniCDx_2037 TGCACAGCCTGAATTTTGCAC Primer (SEQ ID NO: 40) PCR  VIM-S3-CA+TAA+CT+AC+AT+C+C+AC+CCA Blocker LNA2 (SEQ  D NO: 34) /5SpC3/-5′ C3Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ FamFlourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ FlourescentQuencher ™, /3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green topink spectral range, “+”-Locked Nucleic Acid base, “rA”-ribonucleotidebase riboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

TABLE 22  Methylation Primers for VIM_S3_Bottom Strandusing Taqman Probe version “A” Primer Primer Type Name Sequences PCRiCDx_ GTCGAGTTTTAGTCGGAGTTACGTGrATTAA/ 2081 3SpC3/ (SEQ ID NO: 92) PCRiCDx_ GGTGTCGTGGGAAAACGAAACGTAAAAACTACGA 2082CTAArUACTG/3SpC3/ (SEQ ID NO: 93) LDR iCDx_TGGATCGAGACGGAATGCAACCGAGTTTTAGTCG 2083 GAGTTACGTGATCACrGTTCG/3SpC3/ (SEQ ID NO: 94) LDR iCDx_ /5Phos/GTTTATTCGTATTTATAGTTTGGGTAG 2084CGCGTTGCGGTTTCCCTGATTGATACCCGCA  (SEQ ID NO: 98) Taqman iCDx_/5HEX/AGTCGGAGT/ZEN/TACGTGATCACGTT Probe 2085 TATTCGTATTTATAG/3IABkFQ/ (SEQ ID NO: 101) Taqman iCDx_ TGGATCGAGACGGAATGCAAC  Primer 2086(SEQ ID NO: 102) Taqman iCDx_ TGCGGGTATCAATCAGGGAAAC  Primer 2087(SEQ ID NO: 103) PCR  iCDx_ GTT+A+T+GT+G+ATT+A+T+GT+TT+AT+T+ Blocker2088 T+GT+ATTT (SEQ ID NO: 104) /5SpC3/-5′ C3 Spacer, /3SpC3/-3′ C3Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ Fam Flourescent Dye,/5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ Flourescent Quencher ™,/3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green to pink spectralrange, “+”-Locked Nucleic Acid base, “rA”-ribonucleotide baseriboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

TABLE 23 Methylation Primers for VIM_S3_Top Strandusing Taqman Probe version “B” Primer Type Primer Name PCR iCDx_2031GAACTCCAACCGAAACTACGTAArCTACA/ 3SpC3/ (SEQ ID NO: 31) PCR iCDx_2032GGTGTCGTGGACGAGGCGTAGAGGTTGCrG GTTA/3SpC3/ (SEQ ID NO: 32) LDRiCDx_2033A TAGACACGAGCGAGGTCACAACTCCAACCG AAACTACGTAACTGCGrUCCGT/3SpC3/ (SEQ ID NO: 36) LDR iCDx_2034 /5Phos/TCCACCCGCACCTACAACCTAAACAACGCGTGCAAAATTCAGGCTGTGCA  (SEQ ID NO: 37) Taqman iCDx_2035B/5HEX/TTTAACTGC/ZEN/GTCCACCCGC Probe ACCTAC/3IABkFQ/ (SEQ ID NO: 44)Taqman iCDx_2036 TAGACACGAGCGAGGTCAC  Primer (SEQ ID NO: 39) TaqmaniCDx_2037 TGCACAGCCTGAATTTTGCAC  Primer (SEQ ID NO: 40) PCR  VIM-S3-LNA2CA+TAA+CT+AC+AT+C+C+AC+CCA  Blocker (SEQ ID NO: 34) /5SpC3/-5′ C3Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ FamFlourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ FlourescentQuencher ™, /3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green topink spectral range, “+”-Locked Nucleic Acid base, “rA”-ribonucleotidebase riboadenosine; “rT”-ribonucleotide base ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide baseribocytosine

TABLE 24  Methylation Primers for VIM_S3_Bottom Strandusing Taqman Probe version Primer Primer Type Name Sequences PCRiCDx_2081 GTCGAGTTTTAGTCGGAGTTACGTGrATTA A/3SpC3/ (SEQ ID NO: 92) PCRiCDx_2082 GGTGTCGTGGGAAAACGAAACGTAAAAACT ACGACTAArUACTG/3SpC3/ (SEQ ID NO: 93) LDR iCDx_2083 TGGATCGAGACGGAATGCAACCGAGTTTTAGTCGGAGTTACGTGATCACrGTTCG/ 3SpC3/ (SEQ ID NO: 94) LDR iCDx_2084/5Phos/GTTTATTCGTATTTATAGTTTGG GTAGCGCGTTGCGGTTTCCCTGATTGATACCCGCA (SEQ ID NO: 98) Taqman iCDx_2085B /5HEX/TTTGATCAC/ZEN/GTTTATTCGTProbe ATTTATAGTTTGGGTAGCGC/3IABkFQ/  (SEQ ID NO: 99) Taqman iCDx_2086TGGATCGAGACGGAATGCAAC  Primer (SEQ ID NO: 102) Taqman iCDx_2087TGCGGGTATCAATCAGGGAAAC  Primer (SEQ ID NO: 103) PCR  iCDx_2088GTT+A+T+GT+G+ATT+A+T+GT+TT+AT+ Blocker T+T+GT+ATTT (SEQ ID NO: 104)/5SpC3/-5′ C3 Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate,/56-FAM/-5′ Fam Flourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN™ Flourescent Quencher ™, /3IABkFQ/-3′ Iowa Black ® FlourescentQuencher, green to pink spectral range, “+”-Locked Nucleic Acid base,“rA”-ribonucleotide base riboadenosine; “rT”-ribonucleotide ribocytosineribothymidine; “rG”-ribonucleotide base riboguanosine;“rC”-ribonucleotide base

TABLE 25 Results of pixel experiments to detect methylation in HT-29 DNAin the background of Roche hgDNA. GE per Total Templates 130 μl of PCR 12 3 4 5 6 7 8 9 10 11 12 Amplifications HT-29 + 20 GE 21.5 17.5 14.516.0 15.9 14.4 15.0 14.5 14.3 14.2 16.1 13.5 12 Roche (Meth) hgDNA +2500 GE (wt) HT-29 + 10 GE UD 15.2 UD 16.2 UD 15.8 16.3 UD 20.3 16.1 UDUD 6 Roche (Meth) hgDNA + 2500 GE (wt) Roche 2500 GE UD UD UD UD UD 38.5UD 36.8 UD UD 37.6 36.6 0 hgDNA (wt) NTC 0 UD 37.6 UD 37.9 38.3 UD UD UD36.8 38.0 39.5 UD 0

Empirical Example 6 Methylation and Mutation Detection Primer Design

Methylation Assay design was accomplished by first performingcomputerized analysis of the genomic DNA sequence of the Vimentin (VIM)and TMEM90B genes to identify prospective highly methylated CG sites.After the computer analysis and identification of multiple methylationsites, three to four sites within each gene were selected for assaydevelopment. To aid in assay development, a computerized bisulfiteconversion is performed on the bisulfite converted genomic DNA of therespective genes. This is done for both the top and bottom strand todesign oligonucleotides off of the expected bisulfite convertedsequences. The computerized conversion is performed on the top strand byconverting the G bases that are not following an immediate 5′C base intoA bases (G-A Conversion for all G's that are not CG) and for the bottomstrand converting a C base next to an immediate 3′G base into T bases(C-T conversion for all C bases that are not CG). The design criteriafor the ligation assay, starts by identifying the discriminating basethat is either the methylated C or adjacent G (wherein the C of thecomplementary strand is methylated), as the identification of a numberof additional features in the sequence that would aid our designcriteria, including avoiding sequences with very high GC content afterbisulfite conversion. Once the methylated sites for assay developmentare selected, the same site in both the top and bottom strands isassayed. A four step approach was utilized to design oligonucleotidesfor this assay. The first step is to design the upstream and downstreamLigase Detection Reaction (LDR) oligonucleotides. Second, design a genespecific forward and reverse oligonucleotide to cover the LDR site andthe LDR oligonucleotides within a gene-specific amplicon between 100 to140 bases in total length. Third, design fluorescent dyed labeled probes(FAM and HEX™) that cover the ligation site and bind specifically to theligated LDR product. And the final step is to design a corresponding WTLocked Nucleic Acid (LNA™) probe to block amplification of thebisulfite-converted un-methylated promoter region.

(SEQ ID NO: 137) ...AGCCGGCCGAGCTCCAGCCGGAGCTACGT GACTA

GTCCACC CGCAC CTACAGCCTGGGCAG CGCG CTGCGCCCCAGCACCAGCCGCAGCCTCTACGCCTCGTCCCCGGGCGGCGTGTATGCCA CGCG CTCCTCTGC... (SEQ ID NO: 131)...AACCGACCGAACTCCAACCGAAACTACGTAACTACGTCCACCCGCACCTACAACCTAAACAACGCGCTACGCCCCAACACCAACCGCAACCTCTACGCCTCGTCCCCGAACGACGTATATACCACGCGCTCCTCTAC...Top VIM Site 3 (S3) genomic sequence before bisulfite conversion andafter bisulfite conversion. (Red bold nucleotide represents the S3 siteof methylation)

The LDR upstream (Up) oligonucleotide probes are designed with a T_(m)of 64-66° C., with the 3′ end at the discriminating base of interesteither the C or Gin the methylated CG. Once the primer sequence isselected and within the required T_(m) range a technique of blockingnonspecific extension, based off of the IDT (Coralville, Iowa) RNaseH2™cleavage method, is utilized to add a complementary RNA base immediateto the 3′ side of the discriminating base followed by two matched DNAbases at the second and third position and two mismatched DNA bases(preferably either G-T or A-C mismatches) at the fourth and fifth (orthe third and fifth) positions followed by the addition of a 3′ C3Spacer. The overall five base tail and spacer is used to blocknon-specific extension off of the upstream LDR probe. For the upstreamLDR oligonucleotide probe, an additional mismatch (preferably either G-Tor A-C mismatch) is also utilized at the adjacent, second, or thirdposition on the 5′ side of the discriminating base. Additionalmodifications include an optional 5′ C3 Spacer to block lambdaexonuclease digestion of ligation products. After the selection andmodification of the upstream oligonucleotide, a tag-sequencecorresponding to a forward oligonucleotide for a subsequent real-timePCR experiment is added to the 5′ side of the sequence. The tagsequences are chosen from a list of qPCR oligonucleotide forward andreverse primer pairs that were designed previously.

Upstream 

(SEQ ID NO: 132) AACTCCAACCGAAACTACGTAACTGCGrUCCgt/3SpC3/ 

 

 

(SEQ ID NO: 133) TAGACACGAGCGAGGTCACAACTCCAACCGAAACTACGTAACTGCGrUCCgt/3SpC3/

 

 with 5′ Spacer and fourth and fifth  cleavage position mismatch:(SEQ ID NO: 36) /5SpC3/TAGACACGAGCGAGGTCACAACTCCAACCGAAACTACGTAACTGCGrUCCgt/3SpC3/ 

 

 

 with 5′ Spacer and third and fifth  cleavage position mismatch:(SEQ ID NO: 42) /5SpC3/TAGACACGAGCGAGGTCACAACTCCAACCGAAACTACGTAACTGCGrUCtAt/3SpC3/  qPCR Forward Primer:  (SEQ ID NO: 39)5′-TAGACACGAGCGAGGTCAC 3′ (SEQ ID NO: 131). . . AACCGACCGAACTCCAACCGAAACTACGTAACTaCG TCCACCCGCACCTACAACCTAAACAACGCGCTACGCCCCAACACCAACCGCAACCTCTACGCCTCGTCCCCGAACGACGTATATACCACGCGCTCCTCTAC . . . VIM S3 bisulfite converted genomic DNA-Upstream LDR location in Bold,lowercase “a” site of third position mismatch, and the RNA cleavage siteunderlined.

The downstream (Dn) LDR oligonucleotide probe was designed at the baseimmediately following the 3′ side of the discriminating base. Theoligonucleotide probes were designed with a T_(m) of 70-72° C. degrees.Like the upstream probe, a selected tag-sequence is added to the 3′ endof the sequence. Additional modifications made to the design involve theaddition of a mismatch at the fourth position closest to the 5′ side andthe inclusion of additional mismatched bases to the 3′ end of theoligonucleotide right before the real-time tag sequence.

Downstream LDR probe sequence: 

/5Phos/TCCACCCGCACCTACAACCTAAACAA

Downstream LDR probe 

(SEQ ID NO: 37)

5Phos/TCCACCCGCACCTACAACCTAAACAACGCGTGCAAAATTC AGGCTGTGCADownstream oligo with fourth position mismatch and additional basesspacing the bisulfite converted genomic sequence from the qPCR sequence:

(SEQ ID NO: 135) /5Phos/TCCaCCCGCACCTACAACCTAAACAACGCGCTGTGCAAAATTCAGGCTGTGCA qPCR Reverse Primer:  (SEQ ID NO: 40)5′-TGCACAGCCTGAATTTTGCAC-3′ (SEQ ID NO: 131). . . AACCGACCGAACTCCAACCGAAACTACGTAACTACG TCCACCCGCACCTACAACCTAAACAACGCGCTACGCCCCAACACCAACCGCAACCTCTACGCCTCGTCCCCGAACGACGTATATACCACGCGCTCCTCTAC . . . VIM S3 bisulfite converted genomic DNA-Downstream (Dn) LDR location inbold, CG site of methylation is underlined

Gene specific forward (FP) and reverse (RP) oligonucleotide primers weredesigned immediately upstream of the LDR oligonucleotide probes withsignificant overlap of the upstream LDR oligonucleotide probe with atotal amplicon length between about 100-140 bases. The forward andreverse gene-specific oligonucleotide primers were designed with a T_(m)of 62-64° C. The reverse primer was designed further downstream and withno overlap of the Dn LDR oligonucleotide probe, to incorporateadditional CGCG restriction sites when present for furtherdiscrimination of methylated sites. Preferably, one or more Bsh1236I(CGCG) restriction sites are present in the target DNA to allow for anoptional cleaving of un-methylated DNA prior to bisulfite conversion.Optionally, the reverse primer has a non-specific 10 base tail added tothe 5′ end to prevent primer-dimer formation with the other reverseprimers. Both the forward and reverse primers also utilize the RNAcleavage trick at the 3′ end of the primer, but unlike the LDRoligonucleotide probes the primers only have one mismatch at the 3′ endadjacent to the blocking group.

Forward primer:  (SEQ ID NO: 31) 5′-GAACTCCAACCGAAACTACGTAArCTACa/3SpC3/Reverse primer:  (SEQ ID NO: 136) 5′-ACGAGGCGTAGAGGTTGCrGGTTa/3SpC3/Reverse primer + 10 Base Tail (SEQ ID NO: 32) 5′GGTGTCGTGGACGAGGCGTAGAGGTTGCrGGTTA/3SpC3/Forward and Reverse Primer containing the one base mismatch (lowercaseand bold) in the RNA cleavage portion of the primer

(SEQ ID NO: 131) . . . AACCGACCGAACTCCAACCGAAACTACGTAA CTACGTCCACCCGCACCTACAACCTAAACAACGCGCTACGCCCCAACACCAACC GCAACCTCTACGCCTCGTCCCCGAACGACGTATATACCACGCGCTCCTCTAC . . .VIM S3 bisulfite converted genomic DNA-Gene specific Forward (FP) andReverse (RP) Primers in bold, CG site of methylation is enlarged, RNAcleavage site regions are underlined

Of the three to four sites identified by the above analysis two siteswere selected for assay development on both the top and bottom strand.To differentiate between the two sites, strand specific real-time probeswere designed to cover the ligation product with site-specific 5′ FAM-or 5′ HEX™ labeled probes. Real-time probes were designed to incorporatethe third position mismatch that was incorporated into the upstream LDRprobe on the 5′ side of the discriminating base, and an additional probewas designed to cover the 5′ mismatch and the optional fourth positionmismatch on the 3′ side of the discriminating base in the downstreamprobe. The T_(m) of the probes are designed for 68-70° C. Initially, theprobes were designed to have equal coverage of the 5′ and 3′ sides ofthe ligation site, but additional probes were designed with a coveragebias of ⅓ on the (5′) side followed by ⅔ on the downstream side (3′) ofthe ligation site, which was in most cases 7 bases upstream and 15 basesdownstream of the ligation site for most probes. To avoid problems withlow FAM-dye fluorescence when coupled to a G base, all probes weredesigned avoiding a G base at its first 5′ base so dyes can beinterchanged as needed without the need for sequence modification.Additional modifications involve the addition of mismatches immediatelyfollowing the fluorescent dye. The probes were synthesized from IDT andutilize a ZEN™ (IDT) quencher nine bases from the 5′ side, and IowaBlack® FQ (IDT) fluorescent quencher at the 3′ end.

Real-time probe with matching third position mismatch (Bold):

(SEQ ID NO: 38) /5HEX/TAACTGCGT/ZEN/CCACCCGCACCTAC/3IABkFQ/Real-Time Probe With Matching Third Position Mismatch (Bold) and TwoAdditional Bases:

(SEQ ID NO: 44) /5HEX/ttTAACTGC/ZEN/GTCCACCCGCACCTAC/3IABkFQ/(SEQ ID NO: 45) /5HEX/ttTAACTGC/ZEN/GTCCGCCCGCACCTAC/3IABkFQ/(SEQ ID NO: 131) . . . AACCGACCGAACTCCAACCGAAACTACGTAACTaCGTCC A CCCGCACCTACAACCTAAACAACGCGCTACGCCCCAACACCAACCGCAACCTCTACGCCTCGTCCCCGAACGACGTATATACCACGCGCTCCTCTAC . . .VIM S3 bisulfite converted genomic DNA-Site of the Real-time probe,lowercase “a” site of the third position mismatch upstream andunderlined A site of the fourth position mismatch downstream

Locked Nucleic Acid (LNA™) blocking primers were designed to reduceamplification of bisulfite converted un-methylated target, by having aperfectly matched LNA probe binds to the bisulfite convertedun-methylated target and utilizing between 5-10 locked (LNA) bases toprevent amplification. The LNA blocking probes, are synthesized byExiqon (Woburn, Mass.) with a T_(m) of 72-75° C.

TABLE 26 Methylation Detection Oligo List iCDx-2031-VIM-S3-PCR Amplification Forward oligo FP,GAACTCCAACCGAAACTACGTAArCTACA/3SpC3/(SEQ ID NO. 31) iCDx-2032A-VIM-S3- PCR Amplification Reverse oligoRP, GGTGTCGTGGACGAGGCGTAGAGGTTGCrGGTTA/3 SpC3/ (SEQ ID NO. 32)iCDx-2033A-VIM-S3- Upstream LDR oligo with third positionUp, TAGACACGAGCGAGGTCAC mismatch and 4^(th) and 5^(th) cleavage siteAACTCCAACCGAAACT mismatches (bold)ACGTAACTBCGrUCCBT/3SpC3/ (SEQ ID NO. 36) iCDx-2033B-VIM-S3-Up, /5SpC3/TAGACACGAGCGAGGTCAC Upstream LDR oligo with third AACTCCAACCGAAACTACGTAACTGCGrUCCGT/3SpC3/ position and cleavage site(SEQ ID NO. 41) mismatches in 4^(th) and 5^(th)position (bold) and 5' C3 Spacer iCDx-2033D-VIM-S3-Upstream LDR oligo with third Up, /5SpC3/TAGACACGAGCGAGGTCACposition and cleavage site AACTCCAACC mismatches in third andGAAACTACGTAACTGCGrUCTAT/3SpC3/ fifth position(bold) and 5' C3 (SEQ ID NO. 42) Spacer iCDx-2034A-VIM-S3- Downstream LDR oligoDn, /5Phos/TCCACCCGCACCTACAACCTAAACAACGCGTGCAAAATTCAGGCTGTGCA (SEQ ID NO. 37) iCDx-2034D-VIM-S3-Dn, /5Phos/TCCGCCCGCACCTACAACCTAAACAACG Downstream LDR oligo with fourthCGCTGTGCAAAATTCAGGCTGTGCA (SEQ ID NO. 43) position mismatch (bold)iCDx-2035A-VIM-S3-RT-Pb, Real-time probe with matching third/5HEX/TAACTGCGT/ZEN/CCACCCGCACCTAC/ position mismatch (bold)3IABkFQ/ (SEQ. ID NO. 38) iCDx-2035B-VIM-S3-RT-Pb,Real-time probe with matching third/5HEX/ttTAACTGC/ZEN/GTCCACCCGCACCTAC/ position mismatch (bold) and 3IABkFQ/ (SEQ ID NO. 44) pre-sequence mismatches (lowercase)iCDx-2035D-VIM-S3-RT-Pb, Real-time probe with matching5HEX/ttTAACTGC/ZEN/GTCCGCCCGCACCITAC/ third position mismatch 3IABkFQ/ (SEQ ID NO. 45) (Upstream bold) and fourth position(downstream bold) and pre-sequence mismatches (lowercase)iCDx-2036-VIM-S3-RT-FP, Real-time Forward PrimerTAGACACGAGCGAGGTCAC (SEQ ID NO. 39) iCDx-2037-VIM-S3-RT-Real-time Reverse Primer RP, TGCACAGCCTGAATTTTGCAC (SEQ ID NO. 40)VIM-S3-LNA1,  LNA probe (“+” =locked nucleic base)CA + TAA + CT + AC + AT + CC + AC + CCA (SEQ ID NO. 33) VIM-S3-LNA2,LNA probe (“+” =locked nucleic base)CA + TAA + CT + AC + AT + CC + AC + CCA (SEQ ID NO. 34) /5SpC3/ - 5' C3Spacer, /3SpC3/ - 3' C3 Spacer, /5Phos/ - 5' Phosphate, /56-FAM/ -5' Fam Flourescent Dye, /5HEX/ - HEX ™ Fluorescent Dye, /ZEN/ - ZEN™ Flourescent Quencher ™, /3IABkFQ/ - 3' Iowa Black ® FlourescentQuencher, green to pink spectral range, “+” - Locked Nucleic Acid base,“rA” - ribonucleotide base riboadenosine; “rT” - ribonucleotide baseribothymidine; “rG” - ribonucleotide base riboguanosine; “rC” -ribonucleotide base ribocytosine

Mutation detection was performed on a number of different canceroncogenes and tumor suppressors harboring known hot-spot mutationsincluding KRAS (G12A, G12D, G12S, G12C, and G12V), BRAF (V600E,1799T>A), and Tp53 (R248Q, 743G>A). The assay is performed similarly tothe methylation assay using a quantitative Ligase Detection Reactions(LDR) approach except the discriminating base is located at the mutationof interest.

The LDR upstream (Up) oligonucleotide probes are designed with a T_(m)of 64-66° C. 5′ upstream of and ending at the mutation (discriminatingbase of interest). Once the primer is selected off of this base andwithin the required T_(m) range, the technique of blocking nonspecificextension based off of the IDT (Coralville, Ia) RNaseH2™ cleavage methodis utilized to add a complementary RNA base immediate to the 3′ side ofthe discriminating base followed by two matched DNA bases at the secondand third position and two mismatched DNA bases (G-A, C-T) at the fourthand fifth position followed by the addition of a 3′ C3 Spacer. Theoverall five base tail and spacer is used to block non-specificextension off of the upstream LDR probe. For the upstream LDRoligonucleotide probe an additional mismatch (preferably either G-T orA-C mismatch) is also utilized at the adjacent, second, or preferablythird position on the 5′ side of the discriminating base. After theselection and modification of the upstream oligonucleotide, atag-sequence corresponding to a forward primer for a subsequentreal-time PCR experiment is added to the 5′ side of the upstream LDRprobe sequence. The tag sequences are chosen from a list of qPCRoligonucleotide forward and reverse primer pairs that were designedpreviously.

iCDx-308-Br600_(3)-L_Up_Rm: (SEQ ID NO: 4) 5′TAGCGATAGTACCGACAGTCACGtcctAAATAGGTGATTTTGGTCTAG CTACGGArGAAAc/3SpC3/ 

Example of an Upstream LDR Oligo (from 5′: Tag in uppercase and bold,short linker sequence in lowercase italics, upstream LDR probe sequencein uppercase with mismatch in position 3 prior to mutation in bold, andmismatch in last position of five base tail in bold and lowercase)

Additional upstream LDR probes were designed as described above, withthe following modifications: for “A” version LDR probes, the RNA baseimmediate to the 3′ side of the discriminating base followed by threematched DNA bases at the second, third, and fourth position and onemismatched DNA base (G-A, C-T) at the fifth position followed by theaddition of a 3′ C3 Spacer; for “B” version LDR probes, the RNA baseimmediate to the 3′ side of the discriminating base followed by twomatched DNA bases at the second and third position and two mismatchedDNA bases (G-A, C-T) at the fourth and fifth position followed by theaddition of a 3′ C3 Spacer; for “D” versions of the LDR probes, the RNAbase immediate to the 3′ side of the discriminating base followed by twomatched DNA bases at the second and fourth position and two mismatchedDNA bases (G-A, C-T) at the third and fifth position followed by theaddition of a 3′ C3 Spacer. In addition, the “A”, “B” and “D” version ofthe upstream LDR probes had a mismatched DNA base (G-A, C-T) at thethird position from the ligation junction (i.e. 3′ end after cleavagewith RNaseH) and the “D” version of the downstream LDR probes had amismatched DNA base (G-A, C-T) at the fourth position from the ligationjunction (i.e. 5′ end).

The downstream (Dn) LDR oligonucleotide probe was designed at the baseimmediately following the 3′ side of the discriminating base. Theoligonucleotides were designed with a T_(m) of 68° C. degrees. Atag-sequence corresponding to the reverse complement of the reverseprimer for a subsequent real-time PCR experiment is added to the 3′ sideof the LDR specific sequence. Like the upstream LDR oligonucleotideprobe, these sequences were also selected from a predetermined list ofoptimal qPCR primer pairs.

iCDx-276-Br600-L_Dn_P: (SEQ ID NO: 5)/5Phos/GAAATCTCGATGGAGTGGGTCCCATttggt GTGCGGAAACCTA TCGTCGA

Example of a Downstream LDR Oligo (from 5′: upstream LDR probe sequencein uppercase, short linker sequence in lowercase italics, and followedby Tag for real time PCR in uppercase and bold)

Gene-specific forward (FP) and reverse (RP) primers for the PCR stepwere designed immediately upstream or downstream, respectively, of theLDR oligonucleotide probes. There is significant overlap between the FPand the upstream LDR oligonucleotide probe, and there is no overlapbetween the RP and downstream LDR probe. The forward and reversegene-specific oligonucleotide primers were designed with a T_(m) of64-66° C., and with a total amplicon length of 74-94 bases.Additionally, the reverse primer has a non-specific 10 base tail addedto the 5′ end to prevent primer-dimer formation of the downstreamprimers. Optionally, both the forward and reverse primers also utilizethe 5-base RNaseH2™ cleavage method at the 3′ end of the primer, butunlike the LDR oligonucleotide probes, they had zero or one mismatch atthe 3′ end (when that 3′ end is the mutated base to be discriminated, itmatched the wild type sequence and mismatched the mutant sequence).

iCDx-328-Braf_PF_WT_blk2 (gene-specific FP wit 5-base RNaseH2 ™cleavage method and mismatch  at 3′ end):  (SEQ ID NO: 1)CCTCACAGTAAAAATAGGTGATTTTGGTCTArGCTAt/3SpC3/ iCDx-284-Br600-PR (gene-specific RP with 10-base tail in bold, one extra linker base in italics, and gene-specific sequence in uppercase): (SEQ ID NO: 2) ggtgtcgtggTCAAAATGGATCCAGACAACTGTTCAAACExample of a gene-specific forward and reverse PCR primers

For the real time PCR step, strand specific real-time probes labeledwith FAM, HEX™ or TAMRA at the 5′ end were designed to cover theligation product across the junction between the upstream and downstreamLDR primers. Different probes were designed to incorporate either theadjacent, second, or third position mismatch positioned on the 5′ sideof the discriminating base that was incorporated into the correspondingupstream oligos. The probes were designed with a T_(m) of 68° C. Toavoid problems with low FAM dye fluorescence when coupled to a G base,all probes were designed without using a G base at its first 5′ startingbase so dyes can be interchanged as needed without the need for sequencemodification. Additional modifications involve the addition ofmismatches immediately following the fluorescent probe. The probes weresynthesized from IDT and most utilize a ZEN™ quencher nine bases fromthe 5′ side, and Iowa Black® FQ or Iowa Black® RQ (IDT) fluorescentquencher at the 3′ end. iCDx-277_A4: TAGCGATAGTACCGACAGTCAC (SEQ ID NO:6) iCDx-279_C4: TCGACGATAGGTTTCCGCAC (SEQ ID NO: 7)iCDx-281-Br600_(3)_Probe: 5′—/56-TAMN/TA CGG AGA AAT CTC GAT GGA GTGGGT/3IAbRQSp/-3′ (SEQ ID NO: 8)

Example of the Oligos Used in a Real-Time PCR Experiment

Locked Nucleic Acid (LNA™) blocking primers were designed to reduceamplification of wild-type target, as well as ligation of upstream anddownstream LDR probes on amplified wild-type target. To accomplish thisa perfectly matched to Wild-type LNA probe utilizing between 3-6 lockedbases was synthesized by Exiqon (Woburn, Mass.) with a T_(m) of 71-76°C. The LNAs also contain a 5′ phosphate group and a three prime C3Spacer to prevent false-positive results arising from probe extension onWild-type DNA. iCDx-315-BRAF FLW:/5Phos/GCTA+C+AG+T+G+AAAT+CTCG/3SpC3/(SEQ ID NO: 3)

Example of the LNA Oligo Used to Block Wild-Type Signal (LNA Bases areThose Preceded by a + Sign)

Alternatively, Peptide nucleic acid (PNA™) blocking primers weredesigned to reduce amplification of wild-type target, as well asligation of upstream and downstream LDR probes on amplified wild-typetarget. To do this, a perfectly matched to Wild-type PNA oligo wasdesigned to include 4-8 bases on each side of the discriminating base,and to have a Tm of 70-76° C. PNA oligos were synthesized by PNA Bio(Thousand Oaks, Calif.).

(SEQ ID NO: 12) PNA-p53-248-11L: TGAACCGGAGG

Example of a PNA oligo used to block wild-type signal (thediscriminating base is in bold, and it matches the wild-type target)

TABLE 27  Mutation Detection Primer List Step Primer NamePrimer Sequence BRAF Forward Primer iCDx-328-Braf_PF_CCTCACAGTAAAAATAGGTGATTTTGGTCTArG PCR WT_blk2 CTAT/3SpC3/ (SEQ ID NO: 1)Reverse Primer iCDx-284-Br600-PR GGTGTCGTGGTCAAAATGGATCCAGACAACT PCRGTTCAAAC (SEQ ID NO: 2) LNA Blocking iCDx-315-BRAF_FLW/5Phos/GCTA+C+AG+T+G+AAAT+CTCG/ Probe 1 3SpC3/ (SEQ ID NO: 3)Upstream LDR iCDx-308-Br600_(3)- TAGCGATAGTACCGACAGTCACGTCCTAAATAL_Up_Rm GGTGATTTTGGTCTAGCTACGGArGAAAC/ 3SPC3/ (SEQ ID NO: 4)Downstream LDR iCDx-276-Br600- /5Phos/GAAATCTCGATGGAGTGGGTCCCATT L_Dn_PTGGTGTGCGGAAACCTATCGTCGA (SEQ ID NO: 5) Tag Forward iCDx-277_A4TAGCGATAGTACCGACAGTCAC (SEQ ID NO: Primer 6) Tag Reverse  iCDx-279_C4TCGACGATAGGTTTCCGCAC (SEQ ID NO: 7) Primer  Real-Time  iCDx-281-Br600_5′-/56-TAMN/TA CGG AGA AAT CTC GAT Probe (3)_ProbeGGA GTG GGT/3IAbRQSp/-3 (SEQ ID NO: 8) p53 Forward PrimeriCDx-326-p53-248_ CCTGCATGGGCGGCATGrAACCG/3SpC3/ PCR PF_WT_blk2(SEQ ID NO: 9) Reverse Primer iCDx-248-p53-248_PRGGTGTCGTGGAAGTGGCAAGTGGCTCCTGAC PCR (SEQ ID NO: 10) PNA BlockingPNA-p53-248-10 GAACCGGAGG (SEQ ID NO: 11) Probe 1 PNA BlockingPNA-p53-248-11L TGAACCGGAGG (SEQ ID NO: 12) Probe 2 Upstream LDRiCDx-305-P53-248(3)- TCACTATCGGCGTAGTCACCACAGACGCATGG L_Up_RmGCGGCATGAATCArGAGGT (SEQ ID NO: 13) /3SPC3/ Downstream LDRiCDx-202-P53-248-L_ /5Phos/GAGGCCCATCCTCACCATCATCACGTT Dn_PGTTGGTGACTTTACCCGGAGGA (SEQ ID NO: 14) Tag Forward iCDx-82_GTT-GCGC_A2TCACTATCGGCGTAGTCACCA (SEQ ID NO: 15) Primer Tag Reverse  iCDx-244-C2TCCTCCGGGTAAAGTCACCA (SEQ ID NO: 16) Primer  Real-Time iCDx-228-p53-248_ 5′-/56-FAM/CG GCA TGA A/ZEN/T CAG AGG Probe Probe_sCCC ATC C/3IABkFQ/ (SEQ ID NO: 17) KRAS Forward Primer iCDx-327-Kr_12_2_TGACTGAATATAAACTTGTGGTAGTTGGArGC PCR PF_WT_blk2TGG/3SpC3/ (SEQ ID NO: 18) Reverse Primer iCDx-303-Kr-12_GGTGTCGTGGCGTCCACAAAATGATTCTGAAT PCR 1&2_PR TAGCTGTA (SEQ ID NO: 19)PNA Blocking PNA-Kras_12_2-11L GAGCTGGTGGC (SEQ ID NO: 20) Probe 1PNA Blocking PNA-Kras_12_2-11R AGCTGGTGGCG (SEQ ID NO: 21) Probe 2Upstream LDR iCDx-393-Kr-12_ TTCGTACCTCGGCACACCAACATAACTGAATA1(3)-L_Up_Rm TAAACTTGTGGTAGTTGGAGTTHrGTGAT/ 3SpC3/ (SEQ ID NO: 22)Downstream LDR iCDx-222-Kr-12_ /5Phos/GTGGCGTAGGCAAGAGTGCCTTGAC 1-L_Dn_PGGCGTGTGGCTCCGTTACTCTGTCGA (SEQ ID NO: 23) Upstream LDR  iCDx-307-Kr-12_TTCGTACCTCGGCACACCAACATATGAATATA ver-B 2(3)-L_Up_RmAACTTGTGGTAGTTGGAGCCGHrUGGCA/ 3SpC3/ (SEQ ID NO: 27) Upstream LDR iCDx-394-Kr-12_ TTCGTACCTCGGCACACCAACATATGAATATA ver-C 2(3)-L_Up_RmAACTTGTGGTAGTTGGAGCCGHrUGGTA/ 3SpC3/ (SEQ ID NO: 28) Downstream LDRiCDx-269-Kr-12_ /5Phos/TGGCGTAGGCAAGAGTGCCTTGACG ver-B 2-L_Dn_PGCGTGTGGCTCCGTTACTCTGTCGA (SEQ ID NO: 29) Tag Forward iCDx-245_A3TTCGTACCTCGGCACACCA (SEQ ID NO: 24) Primer Tag Reverse  iCDx-246-C3TCGACAGAGTAACGGAGCCA (SEQ ID NO: 25) Primer  Real-Time iCDx-259-T-Kr-5′-/5HEX/TT GGA GTT H/ZEN/GT GGC GTA Probe 1 12_1_ProbeGGC AAG A/3IABkFQ/-3′ (SEQ ID NO: 26) Real-Time  iCDx-270-Kr-12_5′-/5HEX/TAG TTG GAG/ZEN/ CCG HTG GCG Probe 2 2(3)_ProbeTAG G/3IABkFQ/-3′ (SEQ ID NO: 30) /5SpC3/-5′ C3 Spacer, /3SpC3/-3′ C3Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ Fam Flourescent Dye,/5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ Flourescent Quencher ™,/3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green to pink spectralrange, “+”-Locked Nucleic Acid base, “rA”-ribonucleotide baseriboadenosine; “rT”-ribonucleotide ribocytosine ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide base

TABLE 28  Methylation Detection Oligonucleotides Step Name SequenceVIM-S2 Top Strand Forward Primer iCDx-2021-Vim-S2-FPACCACTCTCGCTCCGAAATrCCCCA/3SpC3/  PCR (SEQ ID NO. 46) Reverse PrimeriCDx-2022A-Vim-S2-RP GGTGTCGTGGCGGATTGGTTTTCGGAGAAGAGGrCGA PCRAT/3SpC3/ (SEQ ID NO. 47) Upstream LDR iCDx-2023A-Vim-S2-UpTACCCTCCTAGCTCCGTACATCTCGCTCCGAAATCCTCG ver-ArCGCTG/3SpC3/ (SEQ ID NO: 48) Upstream LDR iCDx-2023B-Vim-S2-Up/5SpC3/TACCCTCCTAGCTCCGTACATCTCGCTCCGAAA ver-BTCCTCGrCGCTG/3SpC3/ (SEQ ID NO. 48) Downstream LDR iCDx-2024A-Vim-S2-Dn/5Phos/CGCCAAAAACGCAACCGCGCTGTGTTGTCTGG ver-A TGGTGCA (SEQ ID NO. 49)Real Time probe iCDx-2025B-Vim-S2-RT-Pb/56-FAM/AT CCT CGC G/ZEN/C CAA AAA CGC ver-B /3IABkF0/ (SEQ ID NO. 50)Real-Time Probe iCDx-2025A-Vim-S2-RT-Pb/56-FAM/CCGAAATCC/ZEN/TCGCGCCAAAAACG/ ver-A 3IABkFQ/ (SEQ ID NO. 51)Tag Forward iCDx-2026-Vim-S2-RT-FP TACCCTCCTAGCTCCGTACA (SEQ ID NO. 52)Primer Tag Reverse iCDx-2027-Vim-S2-RT-RPTGCACCACCAGACAACACA (SEQ ID NO. 53) Primer LNA Blocking VIM-S2-LNA1A+AT+CC+CC+AC+AC+CA+A (SEQ ID NO. 54) Probe 1 LNA Blocking VIM-S2-LNA2A+AT+CC+CC+A+C+AC+CA+A (SEQ ID NO. 55) Probe 2 PNA Blocking VIM-S2-PNA2AATCCCCACACCAAA (SEQ ID NO. 56) Probe 2 VIM-S3 Top Strand Forward PrimeriCDx-2031-VIM-S3-FP GAACTCCAACCGAAACTACGTAArCTACA/3SpC3/ (SEQ PCRID NO. 31) Reverse Primer iCDx-2032A-VIM-S3-RPGGTGTCGTGGACGAGGCGTAGAGGTTGCrGGTTA/ PCR 3SpC3/ (SEQ ID NO. 32)Upstream LDR iCDx-2033A-VIM-S3-Up TAGACACGAGCGAGGTCACAACTCCAACCGAAACTACver-A GTAACTGCGrUCCGT/3SpC3/ (SEQ ID NO. 36) Upstream LDRiCDx-2033B-VIM-S3-Up /5SpC3/TAGACACGAGCGAGGTCACAACTCCAACCGA ver-BAACTACGTAACTGCGrUCCGT/3SpC3/ (SEQ ID NO. 41) Upstream LDRiCDx-2033D-VIM-S3-Up /5SpC3/TAGACACGAGCGAGGTCACAACTCCAACCGAAA ver-DCTACGTAACTGCGrUCTAT/3SpC3/ (SEQ ID NO. 42) Downstream LDRiCDx-2034A-VIM-S3-Dn /5Phos/TCCACCCGCACCTACAACCTAAACAACGCGTG ver-ACAAAATTCAGGCTGTGCA (SEQ ID NO. 37) Downstream LDR iCDx-2034D-VIM-S3-Dn/5Phos/TCCGCCCGCACCTACAACCTAAACAACGCGCT ver-DGTGCAAAATTCAGGCTGTGCA (SEQ ID NO. 43) Real Time probeiCDx-2035B-VIM-S3-RT-Pb /5HEX/TTTAACTGC/ZEN/GTCCACCCGCACCTAC/ ver-B3IABkFQ/ (SEQ ID NO. 44) Real Time Probe iCDx-2035D-VIM-S3-RT-Pb/5HEX/TTTAACTGC/ZEN/GTCCGCCCGCACCTAC/ ver-D 3IABkFQ/ (SEQ ID NO. 45)Real-Time Probe iCDx-2035A-VIM-S3-RT-Pb/5HEX/TAACTGCGT/ZEN/CCACCCGCACCTAC/ ver-A 3IABkFQ/ (SEQ ID NO. 38)Tag Forward iCDx-2036-VIM-S3-RT-FP TAGACACGAGCGAGGTCAC (SEQ ID NO. 39)Primer Tag Reverse iCDx-2037-VIM-S3-RT-RPTGCACAGCCTGAATTTTGCAC (SEQ ID NO. 40) Primer LNA Blocking VIM-S3-LNA1CA+TAA+CT+AC+AT+CC+AC+CCA (SEQ ID NO. 33) Probe 1 LNA BlockingVIM-S3-LNA2 CA+TAA+CT+AC+AT+C+C+AC+CCA (SEQ ID NO. 34) Probe 2PNA Blocking VIM-S3-PNA2 ACATAACTACATCCACCCA (SEQ ID NO. 35) Probe 2TMEM-S1 Bottom Strand Forward Primer iCDx-2051-TMEM90B-REV-GTTAGATATTGGTCGCGGGTTATTATTTGrGACGA/ PCR S1-FP 3SpC3/ (SEQ ID NO. 57)Reverse Primer iCDx-2052-TMEM90B-REV-GGTGTCGTGGGCAACCCGCGCGAAAArTAACT/3SpC3/ PCR S1-RP (SEQ ID NO. 58)Upstream LDR iCDx-2053B-TMEM90B-REV-S1-/5SpC3/TGCTTACCCACGATGCACCCGCGGGTTATTATT ver-B UpTGGAAGCrGATCC/3SpC3/ (SEQ ID NO. 59) Upstream LDRiCDx-2053D-TMEM90B-REV-S1- /5SpC3/TGCTTACCCACGATGCACCCGCGGGTTATTATTver-D Up TGGAAGCrGACTC/3SpC3/ (SEQ ID NO. 60) Upstream LDRiCDx-2053A-TMEM90B-REV-S1- TGCTTACCCACGATGCACCCGCGGGTTATTATTTGGAA ver-AUp GCrGATCC/3SpC3/ (SEQ ID NO. 61) Downstream LDRiCDx-2054D-TMEM90B-REV-S1- /5Phos/GATCTTTCGTTAGGGTTTTTTTGGTTTGGGTTAver-D Dn AAGTTGGTGGGTCGTATGACTTGCTCGCA (SEQ ID NO. 62) Downstream LDRiCDx-2054A-TMEM90B-REV-S1- /5Phos/GATTTTTCGTTAGGGTTTTTTTGGTTTGGGTTAver-A Dn AAGTTGGTCGTATGACTTGCTCGCA (SEQ ID NO. 63) Real Time probeiCDx-2055B-TMEM90B-REV-S1- /56-FAM/AATGGAAGC/ZEN/GATTTTTCGTTAGGGTTTTTTver-B RT-Pb TG/3IABkF0/ (SEQ ID NO: 138) Real Time ProbeiCDx-2055D-TMEM90B-REV-S1- /56-FAM/AATGGAAGC/ZEN/GATCTTTCGTTAGGGTTTTTTver-D RT-Pb TG/3IABkF0/ (SEQ ID NO. 64) Real-Time ProbeiCDx-2055A-TMEM90B-REV-S1- /56-FAM/ATTTGGAAG/ZEN/CGATTTTTCGTTAGGGTTTT/ver-A RT-Pb 3IABkFQ/ (SEQ ID NO. 65) Tag ForwardiCDx-2056-TMEM90B-REV-S1- TGCTTACCCACGATGCACC (SEQ ID NO. 66) PrimerRT-FP Tag Reverse iCDx-2057-TMEM90B-REV-S1-TGCGAGCAAGTCATACGACC (SEQ ID NO. 67) Primer RT-RP LNA BlockingiCDx-2058-TMEM90B-REV-S1- AT+TT+GG+A+T+G+T+GA+TTTTT+T+GTT+AG (SEQ IDProbe 1 LNA NO. 68) TMEM-S3 Bottom Strand Forward PrimeriCDx-2061-TMEM90B-REV- TTTGGGTTGTATTTTGGTGTTTTGTTrATTTA/3SpC3/ PCR S3-FP(SEQ ID NO. 69) Reverse Primer iCDx-2062-TMEM90B-REV-S3-GGTGTCGTGGCTAACTCCGCTACGCTCTCAArUTCTA/ PCR RP 3SpC3/ (SEQ ID NO. 70)Upstream LDR iCDx-2063B-TMEM90B-REV-S3-/5SpC3/TTCGCCTACCGCAGTGAACTGGGTTGTATTTT ver-B UpGGTGTTTTGTTATCTCrGGGGA/3SpC3/ (SEQ ID NO. 71) Upstream LDRiCDx-2063D-TMEM90B-REV-S3- /5SpC3/TTCGCCTACCGCAGTGAACTGGGTTGTATTTT ver-DUp GGTGTTTTGTTATCTCrGGAAA/3SpC3/ (SEQ ID NO. 72) Upstream LDRiCDx-2063A-TMEM90B-REV-S3- TTCGCCTACCGCAGTGAACTGGGTTGTATTTTGGTGTTT ver-AUp TGTTATCTCrGGGGA/3SpC3/ (SEQ ID NO. 73) Downstream LDRiCDx-2064D-TMEM90B-REV-S3- /5Phos/GGGTGACGCGAAGGGGTTGTTGTGCGAAGTT ver-DDn GAGACATGGGCTCGCA (SEQ ID NO. 74) Downstream LDRiCDx-2064A-TMEM90B-REV-S3- /5Phos/GGGAGACGCGAAGGGGTTGTTGTGGTTGAGA ver-ADn CATGGGCTCGCA (SEQ ID NO. 75) Real Time probeiCDx-2065B-TMEM90B-REV-S3- /5HEX/AATTATCTC/ZEN/GGGAGACGCGAAGGG/ ver-BRT-Pb 3IABkFQ/ (SEQ ID NO. 76) Real Time ProbeiCDx-2065D-TMEM90B-REV-S3- /5HEX/AATTATCTC/ZEN/GGGTGACGCGAAGGG/ ver-DRT-Pb 3IABkFQ/ (SEQ ID NO. 77) Real-Time ProbeiCDx-2065A-TMEM90B-REV-S3- /5HEX/TTTTGTTAT/ZEN/CTCGGGAGACGCGAAGGG/ ver-ART-Pb 3IABkFQ/ (SEQ ID NO. 78) Tag Forward iCDx-2066-TMEM90B-REV-S3-TTCGCCTACCGCAGTGAAC (SEQ ID NO. 79) Primer RT-FP Tag ReverseiCDx-2067-TMEM90B-REV-S3- TGCGAGCCCATGTCTCAAC (SEQ ID NO. 80) PrimerRT-RP LNA Blocking iCDx-2068-TMEM90B-REV-S3-TTGTTATT+T+T+GGGAGA+T+G+T+GAAG (SEQ ID NO. Probe 1 LNA 81) VIM-S2 BottomStrand Forward Primer iCDx-2071-VIM-REV-S2-FPGGTTTAGTTTTTTGTTATTTTCGTTTCGAGGrUTTTA/ PCR 3SpC3/ (SEQ ID NO. 82)Reverse Primer iCDx-2072-VIM-REV-S2-RPGGTGTCGTGGGACGATAACGCGAACTAACTCrCCGAG PCR /3SpC3/ (SEQ ID NO. 83)Upstream LDR iCDx-2073B-VIM-REV-S2-Up/5SpC3/TTGCAACAGGCTACCGACCGTTTTTTGTTATTT ver-BTCGTTTCGAGGTTCTCrGCGCC/3SpC3/ (SEQ ID NO. 84) Upstream LDRiCDx-2073A-VIM-REV-S2-Up TTGCAACAGGCTACCGACCGTTTTTTGTTATTTTCGTTT ver-ACGAGGTTCTCrGCGCC/3SpC3/ (SEQ ID NO. 85) Downstream LDRiCDx-2074-VIM-REV-S2-Dn /5Phos/GCGTTAGAGACGTAGTCGCGTTTTATTATTTATATTTATCGCGGGTAGGTAAGGAAGTCACGCA (SEQ ID NO. 86) Real Time probeiCDx-2075B-VIM-REV-S2-RT- /56-FAM/AG GTT CTC G/ZEN/C GTT AGA GAC GTAver-B Pb GTC G/3IABkFQ/ (SEQ ID NO. 87) Real-Time ProbeiCDx-2075A-VIM-REV-S2-RT- /56-FAM/ATTTTCGTT/ZEN/TCGAGGTTCTCGCGTTAGAGAver-A Pb /3IABkFQ/ (SEQ ID NO. 88) Tag ForwardiCDx-2076-VIM-REV-S2-RT-FP TTGCAACAGGCTACCGACC (SEQ ID NO. 89) PrimerTag Reverse iCDx-2077-VIM-REV-S2-RT-RPTGCGTGACTTCCTTACCTACC (SEQ ID NO. 90) Primer LNA BlockingiCDx-2078-VIM-REV-S2-LNA TTT+T+GAG+GTTTT+T+G+T+GTTAGAGA+T+GTA (SEQProbe 1 ID NO. 91_ VIM-S3 Bottom Strand Forward PrimeriCDx-2081-VIM-REV-S3-FP GTCGAGTTTTAGTCGGAGTTACGTGrATTAA/3SpC3/ PCR(SEQ ID NO. 92) Reverse Primer iCDx-2082-VIM-REV-S3-RPGGTGTCGTGGGAAAACGAAACGTAAAAACTACGACTA PCR ArUACTG/3SpC3/ (SEQ ID NO. 93)Upstream LDR iCDx-2083B-VIM-REV-S3-Up/5SpC3/TGGATCGAGACGGAATGCAACCGAGTTTTAGT ver-BCGGAGTTACGTGATCACrGTTCG/3SpC3/ (SEQ ID NO. 94) Upstream LDRiCDx-2083D-VIM-REV-S3-Up /5SpC3/TGGATCGAGACGGAATGCAACCGAGTTTTAGT ver-DCGGAGTTACGTGATCACrGTCTG/3SpC3/ (SEQ ID NO. 95) Upstream LDRiCDx-2083A-VIM-REV-S3-Up TGGATCGAGACGGAATGCAACCGAGTTTTAGTCGGAG ver-ATTACGTGATCACrGTTCG/3SpC3/ (SEQ ID NO. 96)/5Phos/GTTCATTCGTATTTATAGTTTGGGTAGCGCGTT Downstream LDRiCDx-2084D-VIM-REV-S3-Dn GCGTTTTGTTTCCCTGATTGATACCCGCA (SEQ ID NO. ver-D97) Downstream LDR iCDx-2084A-VIM-REV-S3-Dn/5Phos/GTTTATTCGTATTTATAGTTTGGGTAGCGCGTT ver-AGCGGTTTCCCTGATTGATACCCGCA (SEQ ID NO. 98) Real Time probeiCDx-2085B-VIM-REV-S3-RT-Pb /5HEX/TTTGATCAC/ZEN/GTTTATTCGTATTTATAGTTTver-B GGGTAGCGC/3IABkFQ/ (SEQ ID NO. 99) Real Time ProbeiCDx-2085D-VIM-REV-S3-RT-Pb /5HEX/TTTGATCAC/ZEN/GTTCATTCGTATTTATAGTTTver-D GGGTAGCGC/3IABkFQ/ (SEQ ID NO. 100) Real-Time ProbeiCDx-2085A-VIM-REV-S3-RT-Pb /5HEX/AGTCGGAGT/ZEN/TACGTGATCACGTTTATTCGver-A TATTTATAG/3IABkFQ/ (SEQ ID NO. 101) Tag ForwardiCDx-2086-VIM-REV-S3-RT-FP TGGATCGAGACGGAATGCAAC (SEQ ID NO. 102) PrimerTag Reverse iCDx-2087-VIM-REV-S3-RT-RPTGCGGGTATCAATCAGGGAAAC (SEQ ID NO. 103) Primer LNA BlockingiCDx-2088-VIM-REV-S3-LNA GTT+A+T+GT+G+ATT+A+T+GT+TT+AT+T+T+GT+ATTTProbe 1 (SEQ ID NO. 104) TMEM90B-S1 Top Strand Forward PrimeriCDx-2101-TMEM90B-F-S1-FP CTAACCGCGAACCACCATCTAArACGCT/3SpC3/ (SEQ PCRID NO. 105) Reverse Primer iCDx-2102-TMEM90B-F-S1-RPGGTGTCGTGGTTCGCGCGAAGGTGGTTArUTAAC/ PCR 3SpC3/ (SEQ ID NO. 106)Upstream LDR iCDx-2103B-TMEM90B-F-S1-Up/5SpC3/TTGCATTTCGTTAGCGACACAGCGAACCACCA ver-BTCTAAACACGrATCTT/3SpC3/ (SEQ ID NO. 107) Upstream LDRiCDx-2103D-TMEM90B-F-S1-Up /5SpC3/TTGCATTTCGTTAGCGACACAGCGAACCACCA ver-DTCTAAACACGrATTCT/3SpC3/ (SEQ ID NO. 108) Upstream LDRiCDx-2103A-TMEM90B-F-S1-Up TTGCATTTCGTTAGCGACACAGCGAACCACCATCTAAA ver-ACACGrATCTT/3SpC3/ (SEQ ID NO. 109) Downstream LDRiCDx-2104D-TMEM90B-F-S1-Dn /5Phos/ATCTCCCGCTAAAACCTCCCTAATCTAAACCAAver-D AATTAATGTGAGTCGATCTACCCGCA (SEQ ID NO. 110) Downstream LDRiCDx-2104A-TMEM90B-F-S1-Dn /5Phos/ATCCCCCGCTAAAACCTCCCTAATCTAAACCTGver-A TGAGTCGATCTACCCGCA (SEQ ID NO. 111) Real Time probeiCDx-2105B-TMEM90B-F-S1-RT- /56-FAM/AAAAACACG/ZEN/ATCCCCCGCTAAAACCT/ver-B Pb 3IABkFQ/ (SEQ ID NO. 112) Real Time ProbeiCDx-2105D-TMEM90B-F-S1-RT- /56-FAM/AAAAACACG/ZEN/ATCTCCCGCTAAAACCTCC/ver-D Pb 3IABkFQ/ (SEQ ID NO. 113) Real-Time ProbeiCDx-2105A-TMEM90B-F-S1-RT- /56-FAM/CATCTAAAC/ZEN/ACGATCCCCCGCTAA/ ver-APb 3IABkFQ/ (SEQ ID NO. 114) Tag Forward iCDx-2106-TMEM90B-F-S1-RT-TTGCATTTCGTTAGCGACACA (SEQ ID NO. 115) Primer FP Tag ReverseiCDx-2107-TMEM90B-F-S1-RT- TGCGGGTAGATCGACTCACA (SEQ ID NO. 116) PrimerRP LNA Blocking iCDx-2108-TMEM90B-F-S1-LNA CTAAA+C+A+C+A+ATCCCC+C+A+CT Probe 1 (SEQ ID NO. 117) TMEM90B-53 Top Strand Forward PrimeriCDx-2111-TMEM90B-F-53-FP CTAACCTAAACTACACCTTAATACCTTACCArCCCCT/ PCR3SpC3/ (SED ID NO. 118) Reverse Primer iCDx-2112-TMEM90B-F-53-RPGGTGTCGTGGTTTTGTTGGGTAGTTTGGTTTCGTTArCG PCR TTC/3SpC3/ (SEQ ID NO. 119)Upstream LDR iCDx-2113B-TMEM90B-F-53-Up/5SpC3/TGGATCGAGACGGAATGCAACCTACACCTTAA ver-BTACCTTACCACCTCGrAAAGG/3SpC3/ (SEQ ID NO. 120) Upstream LDRiCDx-2113D-TMEM90B-F-53-Up /5SpC3/TGGATCGAGACGGAATGCAACCTACACCTTAA ver-DTACCTTACCACCTCGrAAGAG/3SpC3/ (SEQ ID NO. 121) Upstream LDRiCDx-2113A-TMEM90B-F-53-Up TGGATCGAGACGGAATGCAACCTACACCTTAATACCTT ver-AACCACCTCGrAAAGG/3SpC3/ (SEQ ID NO. 122) Downstream LDRiCDx-2114D-TMEM90B-F-53-Dn /5Phos/AAAGACGCGAAAAAACTACTATACGAATTCGA ver-DTAAAAACTAATAAAACCGAAAACTGTTTCCCTGATTGA TACCCGCA (SEQ ID NO. 123)Downstream LDR iCDx-2114A-TMEM90B-F-53-Dn/5Phos/AAAAACGCGAAAAAACTACTATACGAATTCGA ver-ATAAAAACTAATAAAACCGTTTCCCTGATTGATACCCGC A (SEQ ID NO. 124)Real Time probe iCDx-2115B-TMEM90B-F-53-RT-/5HEX/TTCACCTCG/ZEN/AAAAACGCGAAAAAACTACT/ ver-B Pb3IABkFQ/ (SEQ ID NO. 125) Real Time Probe iCDx-2115D-TMEM90B-F-53-RT-/5HEX/TTCACCTCG/ZEN/AAAGACGCGAAAAAACTAC ver-D PbTATACG/3IABkFQ/ (SEQ ID NO. 126) Real-Time ProbeiCDx-2115A-TMEM90B-F-53-RT- /5HEX/CCTTACCAC/ZEN/CTCGAAAAACGCGAA/3IABkver-A Pb FQ/ (SEQ ID NO. 127) Tag Forward iCDx-2116-TMEM90B-F-53-RT-TGGATCGAGACGGAATGCAAC (SEQ ID NO. 128) Primer FP Tag ReverseiCDx-2117-TMEM90B-F-53-RT- TGCGGGTATCAATCAGGGAAAC (SEQ ID NO. 129)Primer RP LNA Blocking iCDx-2118-TMEM90B-F-S3-LNAACCACCC+C+A+AAAAA+C+A+C+AA  Probe 1 (SEQ ID NO. 130) /5SpC3/-5′ C3Spacer, /3SpC3/-3′ C3 Spacer, /5Phos/-5′ Phosphate, /56-FAM/-5′ FamFlourescent Dye, /5HEX/-HEX ™ Fluorescent Dye, /ZEN/-ZEN ™ FlourescentQuencher ™, /3IABkFQ/-3′ Iowa Black ® Flourescent Quencher, green topink spectral range, “+”-Locked Nucleic Acid base, “rA”-ribonucleotidebase riboadenosine; “rT”-ribonucleotide ribocytosine ribothymidine;“rG”-ribonucleotide base riboguanosine; “rC”-ribonucleotide base

What is claimed is:
 1. A method for identifying in a sample, one or moretarget ribonucleic acid molecules differing in sequence from otherribonucleic acid molecules in the sample due to alternative splicing,alternative transcript, alternative start site, alternative codingsequence, alternative non-coding sequence, exon insertion, exondeletion, intron insertion, translocation, mutation, or otherrearrangement at the genome level, said method comprising: providing asample containing one or more target ribonucleic acid moleculespotentially differing in sequence from other ribonucleic acid molecules;contacting the sample with one or more enzymes capable of digesting dUcontaining nucleic acid molecules potentially present in the sample;providing one or more oligonucleotide primers, each primer beingcomplementary to the one or more target ribonucleic acid molecules;blending the contacted sample, the one or more oligonucleotide primers,a deoxynucleotide mix including dUTP, and a reverse-transcriptase toform a reverse-transcription mixture; generating complementarydeoxyribonucleic acid (cDNA) molecules in the reverse transcriptionmixture, each cDNA molecule comprising a nucleotide sequence that iscomplementary to the target ribonucleic acid molecule and contains dU;providing one or more oligonucleotide primer sets, each primer setcomprising (a) a first oligonucleotide primer comprising a nucleotidesequence that is complementary to a portion of a cDNA nucleotidesequence adjacent to the target ribonucleic acid molecule sequencecomplement of the cDNA, and (b) a second oligonucleotide primercomprising a nucleotide sequence that is complementary to a portion ofan extension product formed from the first oligonucleotide primer;blending the reverse transcription mixture containing the cDNAmolecules, the one or more oligonucleotide primer sets, adeoxynucleotide mix including dUTP, and a polymerase to form apolymerase reaction mixture; subjecting the polymerase chain reactionmixture to one or more polymerase chain reaction cycles comprising adenaturation treatment, a hybridization treatment, and an extensiontreatment thereby forming one or more different primary extensionproducts; providing one or more oligonucleotide probe sets, each probeset comprising (a) a first oligonucleotide probe having a 5′primer-specific portion and a 3′ target sequence-specific portion, and(b) a second oligonucleotide probe having a 5′ target sequence-specificportion and a 3′ primer-specific portion, wherein the first and secondoligonucleotide probes of a probe set are configured to hybridize, in abase specific manner, on complementary portions of a primary extensionproduct corresponding to the target ribonucleic acid molecule sequence;contacting the primary extension products with a ligase and the one ormore oligonucleotide probe sets to form a ligation reaction mixture;subjecting the ligation reaction mixture to one or more ligationreaction cycles whereby the first and second probes of the one or moreoligonucleotide probe sets are ligated together to form ligated productsequences in the ligase reaction mixture, wherein each ligated productsequence comprises the 5′ primer-specific portion, the target-specificportions, and the 3′ primer-specific portion; providing one or moresecondary oligonucleotide primer sets, each secondary oligonucleotideprimer set comprising (a) a first secondary oligonucleotide primercomprising the same nucleotide sequence as the 5′ primer-specificportion of the ligated product sequence and (b) a second secondaryoligonucleotide primer comprising a nucleotide sequence that iscomplementary to the 3′ primer-specific portion of the ligated productsequence; blending the ligated product sequences, the one or moresecondary oligonucleotide primer sets with one or more enzymes capableof digesting deoxyuracil (dU) containing nucleic acid molecules, adeoxynucleotide mix including dUTP, and a DNA polymerase to form asecond polymerase chain reaction mixture; subjecting the secondpolymerase chain reaction mixture to conditions suitable for digestingdeoxyuracil (dU) containing nucleic acid molecules present in the secondpolymerase chain reaction mixture, and one or more polymerase chainreaction cycles comprising a denaturation treatment, a hybridizationtreatment, and an extension treatment thereby forming secondaryextension products; and detecting and distinguishing the secondaryextension products in the sample thereby identifying the presence of oneor more ribonucleic acid molecules differing in sequence from otherribonucleic acid molecules in the sample due to alternative splicing,alternative transcript, alternative start site, alternative codingsequence, alternative non-coding sequence, exon insertion, exondeletion, intron insertion, translocation, mutation, or otherrearrangement at the genome level.
 2. The method of claim 1, wherein thesecond oligonucleotide probe of the oligonucleotide probe set furthercomprises a unitaq detection portion, thereby forming ligated productsequences comprising the 5′ primer-specific portion, the target-specificportions, the unitaq detection portion, and the 3′ primer-specificportion, said method further comprising: providing one or more unitaqdetection probes, wherein each unitaq detection probe hybridizes to acomplementary unitaq detection portion and said detection probecomprises a quencher molecule and a detectable label that are separatedfrom each other; adding the one or more unitaq detection probes to thesecond polymerase chain reaction mixture; and hybridizing the one ormore unitaq detection probes to complementary unitaq detection portionson the ligated product sequence or complement thereof during saidsubjecting the second polymerase chain reaction mixture to conditionssuitable for one or more polymerase chain reaction cycles, whereby thequencher molecule and the detectable label are cleaved from the one ormore unitaq detection probes during the extension treatment, wherebysaid detecting involves the detection of the cleaved detectable label.3. The method of claim 1, said method further comprising: providing oneor more oligonucleotide detection probes, wherein each oligonucleotidedetection probe hybridizes to a ligation product junction portion or itscomplement, and said detection probe comprises a quencher molecule and adetectable label that are separated from each other; adding the one ormore oligonucleotide detection probes to the second polymerase chainreaction mixture; and hybridizing the one or more oligonucleotidedetection probes to complementary detection portions on the ligatedproduct sequence or complement thereof during said subjecting the secondpolymerase chain reaction mixture to conditions suitable for one or morepolymerase chain reaction cycles, whereby the quencher molecule and thedetectable label are cleaved from the one or more oligonucleotidedetection probes during said extension treatment, whereby said detectinginvolves the detection of the cleaved detectable label.